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BaculoDirect GST Gateway Expression Kit

1. Item Amount Cat no Anti glutathione S transferase 0 5 ml A 5800 rabbit IgG fraction 3 mg ml Anti glutathione S transferase 0 5 ml A 11131 rabbit IgG fraction Alexa Fluor 488 conjugate 2 mg ml Glutathione Transferase Fusion 5 purifications G 21801 Protein Purification Kit Glutathione agarose linked through 10 ml G 2879 sulfur sedimented bead suspension 100 ml G 21800 Purification Columns 10 ml 50 R640 50 polypropylene columns Dynabeads M 280 Sheep anti 2 ml 112 03D Rabbit IgG Dynabeads Protein A 2ml 100 01D Dynabeads Protein G 2ml 100 03D WesternBreeze Chromogenic Kit 1 kit WB7103 Anti Mouse WesternBreeze Chromogenic Kit 1 kit WB7107 Anti Goat WesternBreeze Chromogenic Kit 1 kit WB7105 Anti Rabbit WesternBreeze Chemiluminescent 1 kit WB7104 Kit Anti Mouse WesternBreeze Chemiluminescent 1 kit WB7106 Kit Anti Goat WesternBreeze Chemiluminescent 1 kit WB7108 Kit Anti Rabbit WesternBreeze Blocker Diluent 80 ml each WB7050 part A and B WesternBreeze Wash Solution 16X 2 x 100ml WB7003 Overview Introduction Advantages of the BaculoDirect GST Gateway Kits BaculoDirect N GST Linear DNA Introduction The BaculoDirect GST Gateway Transfection and Expression Kits use Gateway Technology to facilitate direct transfer of the gene of interest into the baculovirus genome in vitro without the need for a
2. Step Action Page 1 Generate an entry clone containing your gene of interest 8 2 Perform the LR recombination reaction between the 9 10 BaculoDirect N GST Linear DNA and an entry clone containing your gene of interest using BaculoDirect GST Gateway Transfection Kit 3 Directly transfect insect cells with recombinant baculovirus 11 16 DNA and collect P1 viral stock Screen for recombinant protein expression if desired 4 Infect insect cells with P1 viral stock to generate a high titer 17 19 viral stock Screen for recombinant protein expression Determine titer of viral stock by plaque assay 20 23 Isolate recombinant viral DNA and analyze by PCR if desired 24 28 Infect insect cells and optimize conditions for recombinant 29 32 protein expression 8 Purify recombinant protein if desired 33 Methods Before Starting Introduction Before you start your experiments you will need to have an entry clone containing your gene of interest cultures of Sf9 or Sf21 cells growing and frozen master stocks available Refer to the guidelines below for more information TM Gateway Entry To recombine your gene of interest into the BaculoDirect N GST Linear DNA Vectors you will need an entry clone containing your gene of interest For your convenience Invitrogen offers a variety of Gateway entry vectors see page viii A selection guide for choosing the most appropriate Gateway entry vector for
3. Ganciclovir 100 mM in deionized water 1 Add 26 mg of ganciclovir powder to 800 ul of deionized water 2 Add 1M NaOH dropwise until the solution reaches pH 12 and the ganciclovir dissolves into solution Add HCI dropwise until the solution reaches pH 11 Bring up the final volume to 1 ml with deionized water Filter sterilize the solution through a 0 2 um filter Dn up M Aliquot the solution into multiple tubes and thaw each aliquot only once Store at 20 C protected from light for up to 6 months Thawed aliquots are stable at 4 C for up to 1 month We recommend setting aside the amount of complete growth medium needed for the experiment requiring ganciclovir selection and adding the appropriate amount of ganciclovir to a final concentration of 100 uM Aliquot the remaining ganciclovir into multiple tubes to reduce the number of freeze thaw cycles Additional ganciclovir may be purchased in powder form from InvivoGen Cat no sud gcv TM Ganciclovir provided with the BaculoDirect GST Transfection and Expression Kits has a concentration of 100 mM and might form a precipitate upon thawing Incubating the ganciclovir at 37 C for 10 minutes and vortexing will redissolve the ganciclovir and eliminate the precipitate If you experience ganciclovir precipitation we recommend diluting the 100 mM stock solution in half with sterile distilled water before refreezing for storage Continued on next page 40
4. DNA PCR Procedure 27 Perform the following protocol to lyse cells and extract the viral DNA 1 Transfer 750 ul of your viral supernatant from Step 2 of Isolating Viral DNA for PCR Analysis page 25 to a fresh 1 5 ml microcentrifuge tube Add 750 ul of cold 4 C 20 PEG 8000 in 1 M NaCl Invert the tube twice to mix and incubate at room temperature for 30 minutes Centrifuge at maximum speed for 10 minutes at room temperature to spin down the virus particles Remove all medium from the pellet Note An additional quick spin may be required to remove trace amounts of medium The pellet may not be visible at this point Add 100 ul of lysis buffer 0 1 Triton X 100 in PBS or TBS to the pellet Carefully wash the sides of the tubes to ensure that all of the viral particles are resuspended Add 10 ul of Proteinase K 5 10 mg ml and mix gently by inverting the tube Incubate at 50 C for 1 hour Add 110 ul of phenol chloroform isoamyl alcohol 25 24 1 and mix gently by inverting the tube Centrifuge at maximum speed for 5 minutes at room temperature Transfer the upper aqueous phase to a fresh microcentrifuge tube Add the following reagents to the aqueous phase 3 M sodium acetate 10 ul Glycogen 2 mg ml 5 ul 100 ethanol 250 ul Incubate tubes at 20 C for at least 20 minutes Centrifuge at maximum speed for 15 minutes at 4 C Wash the pellet with 70 ethanol Centrifuge again and remove all traces of ethan
5. Recipes continued PEG NaCI Solution 20 Polyethylene glycol PEG 8000 1M NaCl 1 Add the following reagents to 80 ml of deionized water PEG 8000 20g NaCl 5 84g 2 Bring the final volume to 100 ml with deionized water 3 Autoclave 20 minutes on liquid cycle 4 While the solution is still warm 55 C swirl carefully to mix thoroughly Bluo gal Follow the guidelines below to prepare a 50 mg ml stock solution of Bluo gal 1 Dissolve the Bluo gal in dimethylformamide or dimethyl sulfoxide DMSO to make a 50 mg ml stock solution Use a glass or polypropylene tube Important Exercise caution when working with dimethylformamide Dispense solutions in a vented chemical hood only 2 Donot filter the stock solution 3 Store at 20 C protected from light 41 Map of BaculoDirect N GST Linear DNA Description Map PpH i ATG BaculoDirect N GST Linear DNA was constructed by homologous recombination between wild type Autographa californica multiple nuclear polyhedrosis virus AcMNPV DNA and a transfer plasmid containing a Gateway cassette see map below After recombination the Gateway cassette replaces the native polyhedrin gene resulting in B galactosidase positive polyhedra negative recombinant virus The modified baculovirus genome is linearized at the Bsu36 I site located at the 5 end of the lacZ gene to produce BaculoDirect N Term Linear DNA The map below shows the Gateway
6. We recommend seeding difficult to visualize 8 x 10 cells per well for a six well plate Too many plaques or complete cell lysis Viral titer not dilute enough Prepare additional dilutions of your viral stock for infection Cells are dead Temperature of the plaquing medium is too high Prepare plaquing medium then place ina 47 C water bath until use Cracks in the agarose overlay Growth medium not completely removed Completely aspirate the growth medium before adding the plaquing medium Any remaining growth medium can interfere with the gelling process Continued on next page 36 Troubleshooting continued Protein The following table lists some potential problems and possible solutions to help Expression you troubleshoot your expression studies We recommend including both positive and negative controls in your experiments to verify that correct reagents and protocols were used and to narrow down potential causes of the problem Problem Possible Cause Solution Very little or no recombinant fusion protein detected but cells are infected and dead Entry clone constructed incorrectly Refer to page 8 of this manual and the instructions in the manual for the specific entry vector you are using for guidelines on constructing your entry clone Insert not in frame with N terminal GST tag Recombination error during LR reaction or presence of a premature s
7. cassette elements of the BaculoDirect N GST Linear DNA The first nucleotide of the BaculoDirect N GST Linear DNA sequence corresponds to the first EcoR I site in Homologous Region 1 hr1 For the complete sequence of Autographa californica nuclear polyhedrosis virus refer to GenBank Accession NC_001623 or Ayres M D et al 1994 Pie 1 0 Ppio GST attR1 HSV1 tk Ty lacZ EA Bsu36 BaculoDirect N GST Linear DNA Gateway Cassette Polyhedrin promoter Ppp bases 4430 4556 Polyhedrin Forward priming site bases 4444 4461 Initiation ATG bases 4577 4579 GST tag bases 4580 5249 attR1 recombination site bases 5265 5362 Herpes simplex virus thymidine kinase gene HSV1 tk bases 5649 6779 c Immediate early promoter Pje 1 0 bases 6808 7359 c p10 promoter Pao bases 7407 7504 Bsu36 linearization site base 7755 lacZ ORF bases 7516 10590 attR2 recombination site bases 10606 10730 c complementary strand 42 Features of the BaculoDirect N GST Linear DNA Features 43 Features of the BaculoDirect N GST Linear DNA Gateway cassettes are described below All features have been functionally tested Feature Benefit Polyhedrin promoter Allows efficient high level expression of your recombinant protein Polyhedrin Forward priming site Allows PCR detection and sequencing of the insert GST tag Allows purification of the recombinant fusion protein by affini
8. or factor Xa between the attL1 recombination site and your gene of interest The LR reaction which facilitates the recombination of your entry clone attL substrate and the BaculoDirect N GST Linear DNA attR substrate to create the recombinant expression baculovirus will leave the protease recognition site intact allowing you to remove the GST tag with the appropriate protease For site specific proteases available from Invitrogen refer to www invitrogen com or contact Technical Support page 45 Troubleshooting Transfection The table below lists some potential problems and possible solutions to help you troubleshoot your transfection and initial protein expression screening experiments Problem Possible Cause Solution Transfected cells are dead but Cellfectin II only control is fine Contamination or cytotoxicity from the LR recombination reaction e Include a no Cellfectin II entry clone only negative control e Use PureLink HiPure Plasmid Prep Kit not a silica based miniprep kit for the purification of the entry clone Transfected cells do not show signs of infection Kinetics of infection are slower than expected e Virus production can take up to a week after transfection Observe cells until 8 or 9 days after infection If no signs of infection appear investigate other possible causes Low transfection efficiency e Use Cellfectin II Reagent that is less than 6 months ol
9. 032 Express Five SFM 1000 ml 10486 025 Penicillin Streptomycin 20 ml 15140 148 Fungizone Antimycotic 20 ml 15290 018 Fetal Bovine Serum Qualified 100 ml 16140 063 Heat Inactivated Easy DNA Kit 15 200 reactions K1800 01 PureLink Genomic DNA Mini Kit 10 preps K1820 00 50 preps K1820 01 250 preps K1820 02 PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 BaculoTiter Assay Kit 30 titers K1270 4 Agarose Gel 40 ml 18300 012 B Gal Staining Kit 1 kit K1465 01 Bluo gal 1g 15519 028 CAT Antiserum 50 ul R902 25 Sterile cell culture grade distilled water 500 ml 15230 162 Proteinase K 100 mg 25530 015 UltraPure Glycogen 100 ul 10814 010 NuPAGE LDS Sample Preparation Buffer 10 ml NP0007 4X 250 ml NP0008 Novex Tris Glycine SDS Sample Buffer 20 ml LC2676 2X Continued on next page vii Accessory Products continued Insect Cells Gateway Entry Vectors viii Invitrogen offers a variety of insect cell lines for protein expression studies We recommend using Sf9 or Sf21 cells to generate high titer viral stocks with the BaculoDirect GST Gateway Expression Kits Once you have generated high titer viral stocks you may use Sf9 Sf21 High Five or Mimic Sf9 cells for protein expression studies For more information refer to www invitrogen com or contact Technical Support page 45 Item Amount Cat no Sf9 Frozen Cells 1ml tube B825 01 1 x 107 cells ml Sf21
10. 2 to each well 4 Place the plates in a sealed plastic bag with moist paper towels to prevent evaporation Incubate at 27 C for 72 hours 5 If you wish to isolate viral DNA proceed to Isolating Viral DNA for PCR Analysis next page If you wish to amplify your viral stock proceed to Isolating Virus for Amplification next page Continued on next page 24 Isolating Virus From a Single Plaque continued Isolating Viral T DNA for PCR Analysis 2 3 Collect 2 ml of medium from each well from Step 4 previous page and transfer to sterile 15 ml tubes Centrifuge the tubes at 3 000 5 000 rpm for 5 minutes to remove cells and large debris Transfer the supernatant to fresh 15 ml tubes Proceed to Analyzing Recombinant Viral DNA next page Isolating Virus for 1 Amplification Incubate the cells for 2 more days At 5 days post infection collect 2 ml of medium from each well and transfer to sterile 15 ml tubes Centrifuge the tubes at 3 000 5 000 rpm for 5 minutes to remove cells and large debris Transfer the supernatant to fresh 15 ml tubes Store 1 ml of the viral clone stock at 80 C as a frozen stock and 1 ml at 4 C as a reserve stock Refer to page 16 for additional storage information Proceed to Preparing a High Titer Viral Stock and Screening for Expression page 17 25 Analyzing Recombinant Viral DNA Introduction Viral DNA Purification Materials Needed You may analyze your recombi
11. 300 ng per tubes in 6 tubes 4 C N GST Linear DNA 10 ul of TE buffer pH 8 0 linearized with Bsu36 I Cellfectin II Reagent 1 mg ml in membrane 125 ul 4 C filtered water pENTR CAT Control 40 pl of 0 5 ng ul vectorin 20 ug 20 C Plasmid TE buffer pH 8 0 Ganciclovir 100 mM in deionized 50 ul 20 C water protected from light Gateway LR Clonase II 40 ul 20 C for Enzyme Mix for up to 6 BaculoDirect Kits months 80 C for long term storage Sf21 Frozen Cells 1 x 10 cells ml in 1ml Liquid 60 complete TNM FH nitrogen 30 FBS 10 DMSO Grace s Insect Cell Sterile filtered medium 500 ml 4 C Culture Medium contains L glutamine protected Unsupplemented from light Accessory Products Additional Products Many of the reagents supplied with the BaculoDirect GST Gateway Transfection and Expression Kits as well as other products suitable for use with the kits are available separately from Invitrogen Ordering information is provided below Product Amount Cat no Gateway LR Clonase II Enzyme Mix for 10 reactions 11791 023 BaculoDirect Cellfectin II Reagent 1ml 10362 100 Grace s Insect Cell Culture Medium 500 ml 11595 030 Unsupplemented Grace s Insect Cell Culture Medium 500 ml 11605 094 Supplemented Sf 900 II SFM 500 ml 10902 096 Sf 900 HI SFM 500 ml 12658 019 Sf 900 Medium 1 3X 100 ml 10967
12. Frozen Cells 1 ml tube B821 01 1 x 10 cells ml High Five Cells 1 ml tube B855 02 3 x 10 cells ml Mimic Sf9 Insect Cells 1 ml tube 12552 014 1 x 107 cells ml A variety of Gateway entry vectors are available from Invitrogen Depending on your application you may choose entry vectors with specific features such as a ribosome binding site RBS For more information on the Gateway cloning technology as well as the features and vector maps of available Gateway entry vectors refer to www invitrogen com or contact Technical Support page 45 Item Amount Cat no pENTR TEV D TOPO Cloning Kit 20 reactions K2535 20 pENTR D TOPO Cloning Kit 20 reactions K2400 20 Note A selection guide for choosing the most appropriate Gateway entry vector for your application can be found on our website at www invitrogen com Gateway Continued on next page Accessory Products continued Detection and Purification of Recombinant Proteins You may use western blot analysis to detect and affinity chromatography on glutathione agarose to purify your recombinant fusion protein that is expressed in frame with the N terminal peptide containing the GST glutathione S transferase tag You may also use Dynabeads complexed with anti GST antibodies to isolate your recombinant protein For more information refer to www invitrogen com or contact Technical Support page 45
13. Selection Ganciclovir is a nucleoside analog used in the BaculoDirect GST Gateway Transfection and Expression Kits to negatively select against non recombinant baculovirus Ganciclovir selection which begins immediately after transfection and continues through infection reduces background levels and eliminates the need for plaque purification Ganciclovir is a nucleoside analog 9 1 3 Dihydroxy 2 propoxymethy guanine that is enzymatically phosphorylated by Herpes Simplex Virus type 1 thymidine kinase HSV1 tk Once phosphorylated the active analog incorporates into DNA and inhibits DNA replication Rubsam et al 1999 Ganciclovir selection has been used in Sf9 cells to purify recombinant viruses that have lost the counter selectable gene marker HSV1 tk due to homologous recombination Godeau et al 1992 In the BaculoDirect GST Gateway Transfection and Expression Kits the HSV1 tk gene is under the control of an immediate early promoter PIE 1 0 which drives synthesis of the first viral transcript produced in infected cells Kovacs et al 1991 Ganciclovir is a hazardous material that may cause harm if ingested inhaled or absorbed through the skin Exercise caution and wear suitable protective clothing gloves and safety goggles while handling solutions containing ganciclovir Before handling ganciclovir review the Material Safety Data Sheet available from our website at www invitrogen com msds or by contac
14. approximately 5 6 mg of bovine liver GST per ml of gel Adding excess free glutathione liberates the GST fragment from the matrix which can then be regenerated by washing with a high salt buffer If you are using another resin follow the manufacturer s instructions Glutathione agarose is available from Invitrogen either as a sedimented bead suspension 10 ml or 100 ml or as a part of the Glutathione Transferase Fusion Protein Purification Kit containing anti GST antibodies and purification columns For ordering information see page ix Invitrogen also offers the highly purified rabbit polyclonal anti GST antibody that can be used to purify GST fusion proteins by immunoprecipitation This highly specific antibody which was generated against a 260 amino acid N terminal fragment of the Schistosoma japonica enzyme expressed in Escherichia coli is also useful for detecting GST fusion proteins on western blots see page 32 The intensely green fluorescent Alexa Fluor 488 conjugate of anti glutathione S transferase is also available for direct detection of GST fusion proteins For ordering information see page ix Your purified GST fusion protein expressed from the recombinant baculovirus BaculoDirect N GST Linear DNA x entry clone does not contain a cleavage site for the removal of the N terminal GST tag However you can engineer your Gateway entry clone to encode a recognition site for a specific protease such as TEV thrombin
15. cells into microcentrifuge tubes and centrifuge Wash cells 2X with PBS to remove traces of serum Assay for expression by Western blot analysis For information on preparing protein samples and detecting expression refer to pages 31 32 Store viral stocks as follows If medium is serum free add serum to 10 Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins Store viral stock at 4 C protected from light Store an aliquot of the viral stock at 80 C Do not store routinely used viral stocks at temperatures below 4 C Repeated freeze thaw cycles can result in a 10 to 100 fold decrease in viral titer Once you have obtained your P1 viral stock you may Amplify the viral stock by infecting Sf9 or Sf21 cells refer to Preparing a High Titer Viral Stock and Screening for Protein Expression page 17 We recommend this procedure to obtain the highest viral titers and optimal results in your expression studies Perform a plaque assay to amplify your viral stock from a single viral clone or to determine the titer of your Pl viral stock refer to Performing a Plaque Assay page 20 16 Preparing a High Titer Viral Stock and Screening for Protein Expression Introduction Materials Needed Note Preparing Cells 17 The P1 viral stock is a low titer stock 1 x 10 to 1 x 10 pfu ml You will infect cells with the P1 stock to generate a high titer P2 stoc
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17. in a six well format from cells harvested 24 to 96 hours post infection Other protocols are suitable 1 Add the viral stock to each well at the desired MOI Include the appropriate controls e g mock infected uninfected cells positive control baculovirus previously characterized recombinant baculoviruses Incubate infected cells at 27 C At the appropriate time e g 24 48 72 96 hours post infection harvest the cells and media and place in a 15 ml tube Gently spin to pellet the cells Transfer the cell medium to a fresh tube For analysis of intracellular protein wash the cells 2X with PBS and resuspend the cells in 2 ml of PBS Remove a 15 pl sample and add 5 ul of 4X SDS PAGE Buffer For analysis of secreted protein remove a 15 ul sample of the cell medium and add 5 ul of 4X SDS PAGE Buffer Note The N terminal GST tag may interfere with the secretion of your recombinant protein We recommend that you verify the expression of protein using samples from both the extracellular medium and the cells lysates Freeze samples at 20 C or boil samples for at least 3 minutes and separate proteins by SDS PAGE Analyzing Recombinant Protein Introduction Polyacrylamide Gel Electrophoresis Detecting Recombinant Proteins Assay for CAT Protein Note You may analyze the expression of your recombinant protein by polyacrylamide gel electrophoresis or by Western blot analysis General information for analy
18. minutes at room temperature in the hood Remove the medium Add 2 5 ml plating medium from step 2b per well Proceed to step 3 Prepare the following solutions in 1 5 ml microcentrifuge tubes for each transfection sample Cellfectin II Reagent in the Transfection Mixture A can be left at room temperature for up to 30 minutes Transfection Mixture A Cellfectin II Reagent 8 ul Grace s Insect Medium Unsupplemented 100 pl without supplements serum or antibiotics Transfection Mixture B LR recombination reaction 10 ul Grace s Insect Medium Unsupplemented 100 pl without supplements FBS or antibiotics Procedure continued on next page Continued on next page Transfecting Sf9 or Sf21 Cells continued Transfection Procedure continued from previous page Procedure 4 continued Combine Transfection Mixture A and Transfection Mixture B Mix gently by tapping the tube and incubate at room temperature for 25 35 minutes After 25 35 minutes incubation add the transfection mix from Step 4 total volume 210 ul dropwise onto the cells from step 2 Repeat for all transfections Note With Cellfectin II you do not have to remove the medium from cells and wash cells prior to adding the DNA lipid complex to cells Incubate the cells in a 27 C incubator for 3 to 5 hours Remove the transfection mixture and replace with 2 ml of complete growth medium e g Grace s Insect Medium Supplemented and 10 FBS with
19. perform the plaque assay see recommended dilutions below Viral Stock Dilution P1 10 10 10 10 10 10 P2 10 10 10 10 107 10 P3 10 10 10 107 10 10 The quality of the cell monolayer is critical for a successful plaque assay Be sure to include a cells only control to assess cell viability contamination and monolayer quality 1 Seed 8 x 10 Sf9 or Sf21 cells per well in 2 ml complete growth medium in a six well plate Use 2 to 3 wells for each viral dilution to be tested see Diluting Virus above Gently tip the plate from side to side 4 6 times to evenly distribute the cells 2 Incubate the cells at 27 C for one hour to allow the cells to fully attach to the bottom of the plate 3 Verify that the cells have attached by inspecting them under an inverted microscope 4 Aspirate the medium from the wells Carefully add 1 ml of each viral dilution dropwise to the appropriately labeled well Be careful not to disturb the monolayer 5 Incubate the cells at 27 C for 1 hour While cells are incubating prepare the Plaquing Medium next page Continued on next page Performing a Plaque Assay continued Preparing the Plaquing Medium Agarose Overlay Plaquing medium a mixture of culture medium and agarose is used to immobilize the infected cells for the plaque assay Prepare plaquing medium immediately before use If you are culturing your cells in Sf 900 II SFM or Sf 900 II
20. site specific recombination system which facilitates the integration of lambda into the E coli chromosome and the switch between the lytic and lysogenic pathways Ptashne 1992 In the Gateway Technology the components of the lambda recombination system are modified to improve the specificity and efficiency of the system Bushman et al 1985 This section provides a brief overview of the Gateway Technology For detailed information refer to the Gateway Technology with TM Clonase II manual part no 25 0749 Lambda integration into the E coli chromosome occurs via intermolecular DNA recombination that is mediated by a mixture of lambda and E coli encoded proteins i e LR Clonase II Enzyme Mix for BaculoDirect The hallmarks of lambda recombination are listed below For more detailed information about lambda recombination see published references and reviews Landy 1989 Ptashne 1992 e Recombination occurs between specific att sites on interacting DNA molecules e Recombination is conservative i e there is no net gain or loss of nucleotides and does not require DNA synthesis The DNA segments flanking the recombination sites are switched such that after recombination the att sites are hybrid sequences comprised of sequences donated by each parental vector For example attP sites are comprised of sequences from attR and attL sites e Strand exchange occurs within a core region that is common to all att
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22. the PCR amplification you may use the Polyhedrin forward primer S AAATGATAACCATCTCGC 3 and a primer of your own design that binds within your gene of interest See page 28 for Polyhedrin forward primer binding site PCR reaction conditions must be optimized The Next Step Once the LR reaction is completed you are ready to directly transfect the recombinant baculovirus DNA into insect cells Proceed to the next section for transfection guidelines 10 Transfecting Sf9 or Sf21 Cells Introduction Cellfectin II Reagent Serum Free Medium Note Preparing and Storing Ganciclovir Important 11 This section provides detailed guidelines for transfecting your LR recombination reaction into Sf9 or Sf21 insect cells Sf21 cells are provided with the BaculoDirect GST Gateway Expression Kits Cellfectin II Reagent is supplied with the BaculoDirect GST Gateway Transfection and Expression Kits for lipid mediated transfection of your insect cells Cellfectin II Reagent is a proprietary liposome formulation of a cationic lipid in membrane filtered water and is ideally suited for the transfection of Sf9 and Sf21 insect cells We recommend using Grace s Insect Cell Culture Medium Unsupplemented however you may use serum free medium during the transfection experiment Note that components in serum free medium may interfere with transfection resulting in a decrease in transfection efficiency
23. the plate Verify that the cells have attached by inspecting them under an inverted microscope Continued on next page Preparing a High Titer Viral Stock and Screening for Protein Expression continued Isolating P2 Viral Stock Galactosidase Staining Screening for Expression 1 Add5ulofthe Pl viral stock to each well Place the plates in a sealed plastic bag with moist paper towels to prevent evaporation Incubate infected cells for 72 hours at 27 C 2 At 72 hours post infection collect 2 ml of medium from each well and transfer to sterile 15 ml tubes Centrifuge the tubes at 3 000 5 000 rpm for 5 minutes to remove debris 3 Transfer the supernatant to fresh 15 ml tubes This is the P2 viral stock Store at 4 C protected from light Refer to page 16 for additional storage information 4 With one set of infected cells proceed to B Galactosidase Staining With the other set of infected cells proceed to Screening for Expression We recommend performing both procedures before scaling up your viral stock and performing expression experiments Because the BaculoDirect Linear DNA contains the lacZ gene you may assay for the presence of non recombinant virus by staining the infected cells for B galactosidase expression Recombinant virus will not stain blue because the gene of interest replaces the lacZ gene after the LR recombination reaction see diagram on page 4 If you see blue stained cells we recomme
24. 0 ul 300 ng of DNA at room temperature and mix the contents Do not vortex or pipette up and down as this will shear the baculovirus DNA and reduce transfection efficiency Component Sample Positive Control BaculoDirect N GST Linear DNA 10 ul intube 10 ul in tube pENTR CAT control 100 ng ul 1 u Entry clone 100 300 ng reaction 1 2 ul 1X TE Buffer pH 8 0 4 5 ul 5 ul Total volume 16 ul 16 ul Note To include a negative control set up a third sample reaction and substitute 4 ul of 1X TE Buffer pH 8 0 for the enzyme mix see step 4 TM TM Remove the LR Clonase II Enzyme Mix for BaculoDirect from 20 C and thaw on ice 2 minutes TM TM Vortex the LR Clonase II Enzyme Mix for BaculoDirect briefly twice 2 seconds each time To each sample above add 4 ul of LR Clonase II Enzyme Mix for BaculoDirect or 4 ul of 1X TE Buffer pH 8 0 if preparing a negative control for a total reaction volume of 20 ul Mix well by tapping the tube several times Do not vortex or pipette up and down as this will shear the baculovirus DNA and reduce transfection efficiency Incubate the reactions at 25 C for 1 hour Note Extending the incubation time up to 18 hours typically increases the efficiency of the LR recombination reaction After incubation you may analyze the LR reaction by PCR Dilute a 2 ul aliquot of Note the LR reaction 200 fold and use 2 ul of the dilution in a 25 ul PCR reaction For
25. 100 uM ganciclovir to each well Addition of antibiotics is optional see page 11 Repeat for all transfections Note Distribute the drops evenly to avoid disturbing the monolayer Place the plates in a sealed plastic bag with moist paper towels to prevent evaporation Incubate the cells at 27 C for 72 hours or until you start to see signs of viral infection 14 Isolating P1 Viral Stock Introduction Materials Needed Characteristics of Infected Cells Budded virus should be released into the medium 72 hours after transfection However depending on transfection efficiency cells may not show all of the signs of viral infection for up to a week Beginning at 72 hours after transfection visually inspect the cells daily for signs of infection see below Once the cells appear infected harvest the virus from the cell culture medium using the procedure below You may also perform an initial screen for expression of your recombinant fusion protein if desired e Transfected insect cells from Step 8 previous page e Inverted microscope e 15 ml tubes Virus infected insect cells typically display the following characteristics as observed from visual inspection using an inverted phase microscope at 250 400X magnification Early Increased cell diameter A 25 50 increase in cell diameter may be first 24 hours seen Increased size of cell Nuclei may appear to fill the cells nuclei Late Cessation of
26. Gateway Expression Kits for the transfection experiment For infection expression studies and general culturing of insect cells you may use any complete growth medium e g Sf 900 II SFM Sf 900 III SFM complete TNM FH or other suitable medium Refer to page 40 for a recipe for complete TNM FH C1 y Se 7 When working with recombinant or wild type viral stocks always maintain o Je separate media bottles for cell culture and for virus work Baculovirus particles can d survive and be maintained in media at 4 C and will contaminate your cell cultures if added to tissue culture plates or flasks during passaging Performing the LR Recombination Reaction Introduction Important LR Clonase II Enzyme Mix for BaculoDirect Materials Needed After you have generated an entry clone using most appropriate Gateway entry vector for your application perform the LR recombination reaction to transfer the gene of interest into the BaculoDirect N GST Linear DNA We recommend that you include the pENTR CAT positive control supplied with the kit in your experiments to help you evaluate your results The LR recombination reaction protocol provided on the next page contains optimized amounts of each reagent To obtain the best possible results follow the protocol exactly as described TM TM TM LR Clonase II Enzyme Mix for BaculoDirect is supplied with the BaculoDirect GST Gateway Expression Kits
27. I Optimal MOI will vary between cell lines and the relative infection kinetics of the virus isolate or clone used A dose response should be established for each virus medium reactor and cell line employed to determine the optimal infection parameters to use for protein expression As a starting point infect cells using an MOI of 5 and 10 Refer to page 19 for an equation to determine how much virus stock to add to obtain a specific MOI e Time course We recommend performing a time course to determine the expression kinetics for your recombinant protein as many proteins may be degraded by cellular proteases released in cell culture Note Maximum expression of secreted proteins is generally observed between 30 72 hours and non secreted proteins between 48 96 hours post infection e Secreted proteins If you cloned your gene of interest with a secretion signal sequence be aware that the N terminal GST tag may interfere with the secretion of your recombinant protein We recommend that you verify the expression of protein using samples from both extracellular medium and cells lysates Continued on next page Expressing Recombinant Protein continued Optimizing Expression Materials Needed Preparing Cells You may perform the following to determine the optimal conditions to use to express your recombinant protein of interest Cell line Infect Sf9 Sf21 High Five or Mimic Sf9 cells at a constant MOI Assay for recombina
28. I SFM prepare Sf 900 Plaquing Medium If you are culturing cells in TNM FH prepare Grace s Plaquing Medium Note This procedure provides instructions to prepare 40 or 50 ml of Sf 900 and Grace s Plaquing Medium respectively You will need 2 ml of Plaquing Medium per well To prepare more Plaquing Medium scale up the volume of reagents used accordingly 1 Meltthe 4 Agarose Gel by placing the bottle in a 70 C water bath for 20 to 30 minutes or heating the agarose in a microwave oven While the 4 agarose gel is melting place the following in the 45 C water bath s Empty sterile 100 ml bottle e Sf 900 Medium 1 3X or Grace s Insect Cell Culture Medium 2X as appropriate 2 Once the 4 agarose gel has liquefied move the agarose gel medium and empty 100 ml bottle to a sterile hood 3 Working quickly prepare the plaquing medium as follow Sf 900 Plaquing Medium Combine 30 ml of Sf 900 Medium 1 3X and 10 ml of the melted 4 Agarose Gel in the empty 100 ml bottle and mix gently Grace s Plaquing Medium Add 20 ml of heat inactivated FBS to the 100 ml bottle of Grace s Insect Medium 2X and mix Combine 25 ml of the Grace s Insect Medium 2X containing serum with 12 5 ml of cell culture grade sterile distilled water and 12 5 ml of the melted 4 Agarose Gel in the empty 100 ml bottle and mix gently 4 Add Bluo gal to a final concentration of 150 ug ml Mix immediately by pipetting up and down Place the bottle i
29. I manual part no 25 0749 This manual is available for downloading from www invitrogen com or by contacting Technical Support page 45 The BaculoDirect GST Gateway Expression System is designed to help you construct a recombinant baculovirus to deliver and express a gene of interest in insect cells Use of this system is geared towards those users who are familiar with the principles of baculovirus expression systems and Gateway Technology We highly recommend that users possess a working knowledge of viral and insect cell culture techniques For more information about the baculovirus life cycle viral structure and laboratory techniques refer to the following published reviews King and Possee 1992 O Reilly et al 1992 and Richardson et al 1995 Before starting baculoviral expression experiments we recommend that users refer to the Insect Cell Lines manual part no 25 0127 for additional information on insect cell culture This manual contains information on e Thawing frozen cells e Maintaining and passaging cells e Freezing cells e Scaling up cell culture TM This manual is provided with the BaculoDirect GST Gateway Expression Kits and is also available from www invitrogen com or by contacting Technical Support page 45 The Gateway Technology Introduction Characteristics of Recombination Reactions att Sites The Gateway Technology is based on the bacteriophage lambda
30. Note If you are already culturing Sf9 or Sf21 cells in Sf 900 II SFM or Sf 900 III SFM you can perform the transfection in Grace s Insect Cell Culture Medium Unsupplemented then easily switch back to Sf 900 II SFM or Sf 900 III SFM after transfection Use of complete growth medium that contains antibiotics and antimycotics in addition to ganciclovir for the last step of the transfection protocol see next page is optional If so desired you can use 100 U ml of penicillin 100 ug ml of streptomycin and 0 25 ug ml of amphotericin B i e Fungizone Antimycotic in this last step see page vii for ordering information We recommend setting aside the amount of complete growth medium needed for the experiment requiring ganciclovir selection and adding the appropriate amount of ganciclovir to a final concentration of 100 uM Aliquot the remaining ganciclovir into multiple tubes to reduce the number of freeze thaw cycles Additional ganciclovir may be purchased in powder form from InvivoGen Cat no sud gcv Refer to page 40 for instructions on reconstituting and storing ganciclovir TM Ganciclovir provided with the BaculoDirect GST Gateway Transfection and Expression Kits has a concentration of 100 mM and might form a precipitate upon thawing Incubating the ganciclovir at 37 C for 10 minutes and vortexing will redissolve the ganciclovir and eliminate the precipitate If you experience ganciclovir precipitation we r
31. TGA TATCATGGAG ATAATTAAAA TGATAACCAT CTCGCAAATA Start of transcription Polyhedrin prom oter 4467 AATAAGTATT TTACTGTTTT CGTAACAGTT TTGTAATAAA AAAACCTATA AATATTCCGG ATTATTCATA 4537 CCGTCCCACC ATCGGGCGCG GATCCCCGGG GGATATCACC ATG Ss aml Pro Arg GGC GCA GGT ACC un Met TAC us Thr Ser Leu Tyr Lys 5244 CCG CGT CCA TGG TCG AAT CAA ACA AGT TIG TAC AAA TG BOB EEE EEE EEE EEE 10709 CAGCTTTCTT GTACAAAGTG GTGATAATI GTCGAAAGAA CATGTTTCAC CACTATTAAT ZT attB2 D TCA AAC ATG TIT attB1 TA ATTAAGATCT GAT Ser Pro Ile TCC CCT ATA AGG GGA TAT Lys Ala Gly AAA GCA GGC mM CEL CIE GST Leu Gly Val CTA GGT GTT GAT CCA CAA Thr ACC GOI ACC TGG TGG Taan CCTTTCC TGGGACCCGG CAAGAACCAA a ee Baculovirus DNA 28 Expressing Recombinant Protein Introduction Positive Control Guidelines for Expression 29 Once you have generated a viral stock of suitable titer e g 1 x 10 pfu ml you are ready to use the viral stock to infect insect cells and assay for expression of your recombinant protein Guidelines for infection and expression are provided below If you generated a high titer stock from the positive control construct pENTR CAT we recommend infecting cells with this viral stock to help determine the optimal MOI for your particular cell line and application Once you have infected cells with the positive control virus the gene enco
32. and initial protein expression screening experiments Problem Possible Cause Solution No or little recombinant fusion protein detected in initial expression screen Entry clone constructed incorrectly Refer to page 8 of this manual and the instructions in the manual for the specific entry vector you are using for guidelines on constructing your entry clone Insert not in frame with the N terminal GST tag Refer to the diagram on page 28 to verify the correct reading frame of the resulting recombinant baculovirus DNA following the LR reaction e Analyze recombinant viral DNA by PCR to confirm correct size and orientation e Sequence PCR product to verify proper reading frame for expression of the GST tag High titer viral stock production The table below lists some potential problems and possible solutions to help you troubleshoot your high titer P2 and P3 viral amplification experiments Problem Possible Cause Solution No sign of enlarged cell diameter 72 hours post infection MOI is lower than 0 01 Transfect 70 confluent cells on a 6 well plate with a dilution series of P1 viral stock and monitor the cells every 24 hours Use the MOI that does not produce any morphological changes within 24 hours in 70 80 of the infected cells Cells are of high passage or passed their logarithmic phase of growth e Cells used for viral amplification should be younger than 20 25 pa
33. arch Invitrogen agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee 10 per clone payable to Invitrogen We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes that such clones incorporate the Gateway Technology and that the purchase of Gateway Clonase from Invitrogen is required for carrying out the Gateway recombinational cloning reaction This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerful technology in their research Use of Invitrogen s Gateway Technology including Gateway clones for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Invitrogen s licensing department at 760 603 7200 48 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Ayres M D Howard S C Kuzio J Lopez Ferber M and Possee R D 1994 The Complete DNA Sequence of Autographa californica Nuclear Polyhedrosis Vir
34. bute the System or the vectors or host strains contained in it to others You may not transfer modified altered or original material from the System to a third party without written notification to and written approval from Invitrogen You may not assign sub license rent lease or otherwise transfer any of the rights or obligations set forth herein except as expressly permitted by Invitrogen 46 Purchaser Notification continued Limited Use Label License No 137 BaculoDirect Expression System Limited Use Label License No 19 Gateway Cloning Products Gateway Clone Distribution Policy 47 This product is the subject of U S Patent No 5 169 784 licensed to Invitrogen Corporation by the Texas A amp M University System For information on purchasing a license to this product for purposes other than research contact The Texas A amp M University System College Station TX 979 847 8682 This product and its use is the subject of one or more of U S Patent Nos 5 888 732 6 143 557 6 171 861 6 270 969 and 6 277 608 and or other pending U S and foreign patent applications owned by Invitrogen Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any meth
35. cell growth Cells appear to stop growing when compared 24 72 hours to a cell only control Detachment Cells release from the plate or flask Very Late Cell lysis Cells appear lysed and there are signs of gt 72 hours clearing in the monolayer Isolating P1 Viral Stock 15 1 Once the transfected cells demonstrate signs of very late stage infection e g 72 hours post transfection collect 2 ml of medium from each well and transfer to sterile 15 ml tubes Centrifuge the tubes at 3000 5000 rpm for 5 minutes to remove cells and large debris 2 Transfer the supernatant to fresh 15 ml tubes This is the P1 viral stock Store at 4 C protected from light See the next page for additional storage information 3 Ifyou wish to screen for expression of your recombinant fusion protein proceed to Screening for Expression next page Continued on next page Isolating P1 Viral Stock continued Screening for Expression Storing Viral Stocks The Next Step You may perform a small scale or preliminary expression experiment on the transfected cells to verify expression of your recombinant protein Follow the general guidelines below to assay for expression If you are expressing a secreted protein remove a sample from the medium to analyze protein expression and secretion You may also harvest cells to analyze intracellular levels of your recombinant protein see below To harvest cells transfer transfected
36. d and do not freeze Cellfectin II Reagent for storage e Perform transfection in Grace s Insect Medium Unsupplemented that does not contain supplements antibiotics or FBS Cells are not viable e Cells should be in log phase and 95 98 viable e Refer to the Insect Cell Lines manual for tips on culturing Sf9 and Sf21 cells Cells are not confluent enough Plate cells at 50 70 confluence Cells are of high passage Use cells that are between 8 to 15 passages Cells are of too low passage After reviving cells grow them for at least 5 passages before transfection Cells are too dense Plate 8 x 10 cells per well for six well plates Split the cells if they are too confluent 3 days after transfection Add complete growth medium containing 100 uM ganciclovir and incubate for 1 to 2 more days at 27 C LR recombination reaction unsuccessful e Make sure you added the LR Clonase II Enzyme Mix for BaculoDirect Kits to the LR reaction e Check the pENTR CAT positive control transfection to verify that the LR reaction was successful e Check your LR reaction using PCR as described on page 10 e Incubate LR reactions for up to 18 hours to increase recombinational efficiency Continued on next page 34 Troubleshooting continued Transfection The table below lists some potential problems and possible solutions to help you continued troubleshoot your transfection
37. dditional cloning or recombination in bacterial or insect cells The resulting recombinant baculovirus DNA is transfected directly into insect cells to generate recombinant virus and to screen for expression The ability to clone and express genes from baculovirus without plaque purification or selection in bacteria makes the BaculoDirect GST Gateway Transfection and Expression Kits the fastest procedure for baculovirus expression Using the BaculoDirect GST Gateway Transfection and Expression Kits to obtain purified recombinant virus suitable for production of high titer stocks offers the following advantages e Saves time by allowing rapid cloning of the gene of interest into the baculovirus genome without the need for traditional homologous recombination or site specific transposition methods e Produces high level expression of a GST tagged recombinant protein for easy detection and purification e Linearized baculovirus DNA and ganciclovir selection inhibits replication of non recombinant virus and eliminates the need for plaque purification e The GST tag is helpful for solubilization of the overexpressed protein of interest as it prevents the fusion protein from being sequestered into inclusion bodies The major features of the BaculoDirect N GST Linear DNA include e attR1 and attR2 sites for recombinational cloning of the gene of interest from a Gateway entry clone e Herpes simplex virus thymidine kinase
38. ding chloramphenicol acetyltransferase CAT will be constitutively expressed and can be easily assayed see page 32 General guidelines are provided below to infect insect cells with the recombinant baculovirus to express your protein of interest s Cell line Depending on your application and gene of interest you may use any insect cell line e g Sf9 Sf21 High Five Mimic Sf9 for expression Cells may be grown in adherent or suspension culture in the culture vessel of choice Note If you are expressing a secreted protein you may improve expression by using High Five cells e Culture Conditions We generally culture cells in serum free conditions using Sf 900 II SFM Sf 900 III SFM or Express Five SFM as appropriate see page vii Depending on your application and the protein of interest note that it may be necessary to supplement the culture post infection with 0 1 to 0 5 FBS or BSA to protect the recombinant protein from proteolysis Protein based protease inhibitors are generally less expensive and more effective than many synthetic protease inhibitors e Infection Conditions We recommend infecting cultures while cells are in the mid logarithmic phase of growth at a density of 1 5 x 10 to 2 5 x 10 cells ml Make sure that the culture is not rate limited by nutritional i e amino acid or carbohydrate utilization or environmental factors i e pH dissolved Oz or temperature during infection e MO
39. ecommend diluting the 100 mM stock solution in half with sterile distilled water before refreezing for storage Continued on next page Transfecting Sf9 or Sf21 Cells continued Materials Needed Controls Important s Sf21 cells provided with the BaculoDirect GST Gateway Expression Kits or Sf9 cells s LR reaction from LR Recombination Reaction Protocol page 10 e Grace s Insect Cell Culture Medium Unsupplemented provided with the BaculoDirect GST Gateway Expression Kits also available separately see page vii e Cellfectin II Reagent provided with the BaculoDirect GST Gateway Transfection and Expression Kits also available separately see page vii e Complete growth medium e g Sf 900 II SFM or other suitable medium with antibiotics and 100 uM ganciclovir see Note on previous page e Six well tissue culture plates e 27 C incubator e Air tight bags or containers e Inverted microscope Note All the reagents and cell lines necessary for transfection are also available separately from Invitrogen For ordering information see page vii viii We recommend that you include the following controls in your experiments e LRrecombination reaction using pPENTR CAT plasmid as a positive control s Cellfectin II Reagent only mock transfection as a negative control Use Grace s Insect Medium Unsupplemented which does not contain any supplements FBS or antibiotics for the transfection pr
40. eded We recommend you perform a plaque assay to determine the titer of your viral stock You may also perform a plaque assay to purify a single viral clone if desired In this procedure you will infect cells with dilutions of your viral stock and identify focal points of infection plaques on an agarose overlay You may also titer your viral stock by the end point dilution method described in O Reilly et al 1992 We recommend using the BaculoTiter Assay Kit available separately from Invitrogen to determine the titer of your baculoviral stock The BaculoTiter Assay Kit rapidly determines the titer of an unknown baculovirus sample with minimal handling steps providing both accuracy and convenience in an easy to use kit format in two days as opposed to ten days with the serial dilution assays See page vii for ordering information You will use a chromogenic substrate to distinguish colorless plaques containing recombinant virus from blue plaques containing non recombinant virus We recommend using Bluo gal instead of X gal for blue white screening because Bluo gal generally produces a darker blue color than X gal Add Bluo gal directly to the overlay solution before pouring over the infected cells s Sf21 cells provided with BaculoDirect GST Gateway Expression Kits or available separately see page viii or Sf9 cells available separately see page viii e Sf 900 II Sf 900 III SFM or other appropriate comple
41. erties of the IE1 and IEO Transactivators Encoded by the Baculovirus Autographa californica Multicapsid Nuclear Polyhedrosis Virus J Virol 65 5281 5288 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 O Reilly D R Miller L K and Luckow V A 1992 Baculovirus Expression Vectors A Laboratory Manual W H Freeman and Company New York N Y Ptashne M 1992 A Genetic Switch Phage Lambda and Higher Organisms Cell Press Cambridge MA 49 References continued Richardson C D ed 1995 Baculovirus Expression Protocols Vol 39 Methods in Molecular Biology Edited by Walker J M Humana Press Totowa NJ Rubsam L A Boucher P D Murphy P J KuKuruga M and Shewach D S 1999 Cytotoxicity and Accumulation of Ganciclovir Triphosphate in Bystander Cells Cocultured with Herpes Simplex Virus Type 1 Thymidine Kinase expressing Human Glioblastoma Cells Cancer Research 59 669 675 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguis
42. gene HSV1 tk located between the two attR sites for negative selection using ganciclovir e lacZ gene located between the two attR sites for determination of viral purity using B galactosidase staining e N terminal GST fusion tag for detection and purification of recombinant fusion proteins Note If you are planning on expressing a secreted protein be aware that the presence of the GST tag may interfere with secretion Continued on next page Overview continued The Gateway Technology Important Insect Cell Lines Manual Gateway Technology is a universal cloning method based on the site specific recombination properties of bacteriophage lambda Landy 1989 The Gateway Technology provides a rapid and highly efficient way to move DNA sequences into multiple vector systems for functional analysis and protein expression To produce recombinant baculovirus using the BaculoDirect GST Gateway Expression Kit simply 1 Clone the gene of interest into a Gateway entry vector of choice to create an entry clone 2 Perform an LR recombination reaction to transfer the gene of interest from the entry clone to the BaculoDirect N GST Linear DNA 3 Transfect insect cells with recombinant baculovirus DNA and harvest recombinant baculovirus For more detailed information about Gateway Technology generating an entry clone and performing the LR recombination reaction refer to the Gateway Technology with Clonase I
43. hes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Weisberg R A and Landy A 1983 in Lambda II Hendrix R W Roberts J W Stahl F W and Weisberg R A eds pp 211 250 Cold Spring Harbor Press Cold Spring Harbor NY 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 8 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
44. iled guidelines for plaque purifying your virus Isolated virus can be used to generate a viral stock from a single viral clone or for PCR analysis of the recombinant baculovirus DNA If you do not wish to plaque purify your virus proceed to Expressing Recombinant Protein page 29 Sf21 cells provided with the BaculoDirect GST Gateway Expression Kits also available separately see page viii or Sf9 cells available separately see page viii e Complete growth medium e Plates containing plaques from Step 5 page 22 e Six well tissue culture plates e 27 C incubator e Inverted microscope e Sterile Pasteur pipette and bulb e Air tight bags or containers Use log phase Sf9 or Sf21 cells with greater than 95 viability 1 Seed 8 x 10 Sf9 or Sf21 cells per well in 2 ml of complete growth medium in a six well tissue culture plate Gently tip the plate from side to side 4 6 times to evenly distribute the cells 2 Incubate the cells at 27 C for one hour to allow the cells to fully attach to the bottom of the plate 3 Verify that the cells have attached by inspecting them under an inverted microscope 1 Using a sterile Pasteur pipette and bulb carefully penetrate and remove the agarose containing the desired plaque 2 Transfer the agarose plug containing the plaque to a 1 5 ml microcentrifuge tube containing 500 ul of complete growth medium Mix well by vortexing Add 100 ul of the agarose plug solution from Step
45. ing time established during small scale test expression as a guideline change harvesting time 6 hours while keeping the MOI constant Viral stock worked well initially but after a couple of months expression levels decreased considerably Viral stock was originally amplified using high MOI Re amplify virus from lower passage stock using low MOI 0 01 0 1 Did not centrifuge and discard cells when harvesting viral supernatant Re amplify virus from lower passage stock using low MOI 0 01 0 1 If this viral stock is P2 it can be used in amplification For certain genes the virus can become very unstable Freeze the aliquoted viral stock and perform one round of amplification after reviving the virus 39 Recipes Complete TNM FH Medium Ganciclovir Stock Solution Ganciclovir Working Solution Important Appendix Complete TNM FH medium is Grace s Insect Medium with supplements lactalbumin hydrolysate L glutamine TC yeastolate and 10 fetal bovine serum FBS 1 If you are using Grace s Insect Medium Supplemented add 55 ml of FBS Mix well 2 To include antibiotics and antimycotics add the following at the recommended concentration Penicillin 100U ml Streptomycin 100 ug ml Amphotericin 0 25 ug ml 3 Filter sterilize the solution through a 0 2 um filter into a sterile container A pre filter may be required 4 Store at 4 C and warm to 27 C before use
46. invitrogen BaculoDirect GST Gateway Expression Kit For cloning and high level expression of recombinant GST fusion proteins using Gateway adapted Baculovirus DNA Catalog nos A10640 A10641 Version A 15 December 2008 A10607 ii Table of Contents Table ot e na a rn Ran TETEE Ei OESE EEEa iii Kit Contents and Storage zss csc csschiasdsabaseesdassaesssagedestsedances sahara ardea a ETa rag SR ee Y Accessory Products TTT vii INroducHonn unsestesneiee ehesten tee 1 Oveiviewinsessiunsskesbtessklibnshbeiliheeilbelhin the in hen la 1 TheGateway CCU Oe set se in 3 Gan Ciclo vars ST ts HT TTT E E R 5 Experimenfal Overview n a sense ea Rhe heine 6 MethodS nasse ee een een eos 8 Before Starting an 2er a cence stebdaseacdesagensahdassth paste RRRR eanmeknlalekneieheheiied ssenikteileitene 8 Performing the LR Recombination Reachion sees eee eee ereer 9 Transfecting S9 or S2 elsi ses iin atten ale ken anna 11 Isolating Pl Viral Stock nassen hen en en Elan 15 Preparing a High Titer Viral Stock and Screening for Protein Expression 17 Performing a Plaque Assay uneeessssnseneeensnnnnnennnnnennnnnsnsnsnsnnnnnnnnnnnnnnnnnnnennnsennsnsnsnnnnnnnnsnsnsnnnnnnnn 20 Analyzing Recombinant Viral DNA eeigen aeieea ee eraan ka a aKT EBE ERE tE EEA R AE neS ROSER ABONE REEERE 26 Expressing Recombinant T PTT 29 Analyzing Recombinant Protein sassseisisinesesteninsniisheenisnauninl none glas 32 Purifying Recombinant Protein een
47. is also available separately from Invitrogen see page vii for ordering information Other commercial kits are available for assaying CAT expression The molecular weight ofthe CAT fusion protein is approximately 53 kDa Due to the presence of the attB sites there will be additional amino acids between your gene of interest and the N terminal GST tag see page 28 for a diagram Expression of your protein with the GST tag will increase the size of your recombinant protein by approximately 27 8 kDa 32 Purifying Recombinant Protein Introduction Glutathione Agarose Anti Glutathione S Transferase Antibody Removal of the GST tag 33 Once you have optimized expression levels you may purify your recombinant GST fusion protein either by affinity chromatography on glutathione agarose or by immunoprecipitation using anti GST antibodies General information for purifying your recombinant GST fusion protein is provided below For detailed instructions consult the literature provided with each product which are also available through our website at www invitrogen com or by contacting Technical Support page 45 Invitrogen offers glutathione agarose for the purification of your recombinant GST fusion protein by affinity chromatography in a single step see page vii for ordering information The glutathione agarose consists of glutathione linked via the sulfur atom to crosslinked beaded agarose and has a binding capacity of
48. k of approximately 5 x 107 to 1 x 10 pfu ml This P2 viral stock can then be used to generate a large scale high titer viral stock suitable for expression studies Guidelines are provided in this section to amplify the recombinant baculovirus and to screen for recombinant protein expression Sf21 cells provided with the BaculoDirect GST Gateway Expression Kits or available separately see page viii or Sf9 cells available separately see page viii for ordering information Complete growth medium with 100 uM ganciclovir P1 viral stock from Step 2 page 15 B Gal Staining Kit recommended see page vii for ordering information or other suitable kit Six well tissue culture plates 27 C incubator Inverted microscope Air tight bags or containers You will infect duplicate samples of Sf9 or Sf21 cells with P1 viral stock One set of cells will be assayed for the presence of non recombinant virus by B galactosidase staining The other set will be assayed for expression of your recombinant protein Use log phase Sf9 or Sf21 cells with greater than 95 viability 1 Seed 8 x 10 Sf9 or Sf21 cells per well in 2 ml of complete growth medium with 100 uM ganciclovir in a six well tissue culture plate Remember to seed duplicate wells see Note above Gently tip the plate from side to side 4 6 times to evenly distribute the cells Incubate the cells at 27 C for one hour to allow the cells to fully attach to the bottom of
49. makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Introduction Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 69 Baculovirus Vectors and Reagents Use of the BaculoDirect GST Gateway Transfection and Expression Kits is covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party
50. ment Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Invitrogen Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 For additional information about Invitrogen s policy for the use and distribution of Gateway clones see the section entitled Gateway Clone Distribution Policy next page Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions The information supplied in this section is intended to provide clarity concerning Invitrogen s policy for the use and distribution of cloned nucleic acid fragments including open reading frames created using Invitrogen s commercially available Gateway Technology Invitrogen understands that Gateway entry clones containing attL1 and attL2 sites may be generated by academic and government researchers for the purpose of scientific research Invitrogen agrees that such clones may be distributed for scientific research by non profit organizations and by for profit organizations without royalty payment to Invitrogen Invitrogen also understands that Gateway expression clones containing attBl and attB2 sites may be generated by academic and government researchers for the purpose of scientific rese
51. n a 47 C water bath until use Pouring the agarose overlay may require some practice if you are unfamiliar with this technique You should already have your plaquing medium prepared see above remove from water bath It is important to work quickly and efficiently 1 After the 1 hour incubation period Step 5 previous page remove the cells from the incubator and completely aspirate the medium from each well containing cells and virus If you have multiple plates follow this protocol for one plate before proceeding to the next Do not let the cells dry out 2 Withdraw 2 ml of the plaquing medium and slowly stream the solution down the side of the well Repeat for all wells Do not move the plate until the agarose overlay has set Repeat Steps 1 2 until all plates have been completed Place the plates in a sealed plastic bag with moist paper towels to prevent evaporation Note Once condensation appears on the plastic bag or container open the bag or container Moisture can destroy the monolayer preventing plaque formation 5 Incubate the cells at 27 C for 4 6 days or until plaques are well formed Proceed to Calculating Viral Titer next page Continued on next page 22 Performing a Plaque Assay continued Neutral Red Overlay Calculating Viral Titer Example The Next Step 23 To improve visualization of plaques you may perform a neutral red overlay 4 days post infection Do not use this procedure if y
52. nant viral DNA by PCR to verify the presence and orientation of your gene of interest You may also use the PCR procedure on the next page to confirm your recombinant baculovirus DNA after the LR reaction We recommend including a negative control no DNA template in your experiments to help you evaluate your results Invitrogen offers a variety of products that enable high yield high purity DNA extraction from a wide range of sample types For fast and easy isolation of DNA from baculoviruses we recommend using the PureLink Genomic DNA Mini Kit or the Easy DNA Kit see page vii for ordering information Follow the protocol provided with the kit manual for isolating baculovirus DNA All Invitrogen manuals are available for downloading from our website www invitrogen com or by contacting Technical Support page 45 An alternative protocol is also provided on the next page to isolate your baculovirus DNA e Viral supernatant from Isolating Viral DNA for PCR Analysis previous page e 20 PEG 8000 in 1 M NaCl at 4 C see page 41 for a recipe e Lysis buffer 0 1 Triton X 100 in PBS or TBS e Proteinase K 5 10 mg ml see page vii for ordering information e Phenol chloroform isoamyl alcohol 25 24 1 e 3Msodium acetate e Glycogen 2 mg ml see page vii for ordering information e 100 ethanol e 70 ethanol e 50 C water bath Continued on next page 26 Analyzing Recombinant Viral DNA continued Isolating Viral
53. nd that you perform a plaque assay to isolate a recombinant viral clone see page 20 You will need to verify expression of your recombinant protein before further amplifying your viral stock Follow the general guidelines below to assay for expression e Ifyou are expressing a secreted protein remove a sample from the medium to analyze protein expression and secretion You may also harvest cells to analyze intracellular levels of your recombinant protein see below Note The presence of the GST tag on your recombinant protein may interfere with its secretion e To harvest cells transfer transfected cells into microcentrifuge tubes and centrifuge to collect cells Wash cells 2X with PBS to remove traces of serum e Assay for expression by western blot analysis For information on preparing protein samples and detecting expression refer to pages 31 32 Continued on next page 18 Preparing a High Titer Viral Stock and Screening for Protein Expression continued Scaling Up the Amplification Procedure Multiplicity of Infection MOI Generating High Titer Stocks From Frozen Master Stock The Next Step 19 If you are satisfied with the purity of the viral stock and have confirmed expression of your recombinant protein you may scale up the amplification procedure to any volume of your choice To produce a large scale high titer P3 stock we recommend doing the following e Performa plaque assay to determine
54. nt protein expression at different times post infection e g 24 48 72 96 hours post infection Choose the cell line that provides the optimal level of recombinant protein expression MOI Infect a population of cells at varying MOIS e g 1 2 5 10 20 and assay for protein expression Use the MOI that provides the optimal level of recombinant protein expression Time course Infect cells at a constant MOI and assay for recombinant protein expression at different times post infection e g 24 48 72 96 hours post infection Choose the time point at which optimal recombinant protein expression is obtained Insect cells of choice Complete growth medium Viral stock of known titer gt 1 x 10 pfu ml SDS PAGE Loading Buffer NuPAGE LDS Sample Buffer or Novex Tris Glycine SDS Sample Buffer see page vii Six well tissue culture plate 27 C incubator Inverted microscope Seed 1x 10 2 x 10 insect cells per well in 2 ml complete growth medium in a six well tissue culture plate Incubate the cells at 27 C for one hour to allow the cells to fully attach to the bottom of the plate Verify that the cells have attached by inspecting them under an inverted microscope Continued on next page 30 Expressing Recombinant Protein continued Preparing Protein Samples 31 Use the following procedure to prepare samples of your recombinant protein This procedure is designed to allow expression analysis
55. ocedure The proteins in the FBS and supplements will interfere with the Cellfectin II Reagent causing the transfection efficiency to decrease Continued on next page 12 Transfecting Sf9 or Sf21 Cells continued Transfection Procedure 13 For Sf9 or Sf21 insect cells cultured in Grace s Insect Medium Supplemented containing 10 FBS use the following protocol to transfect your cells in a 6 well format All amounts and volumes are given on a per well basis Verify that the Sf9 or Sf21 cells are in the log phase 1 5 2 5 x 10 cells ml with greater than 95 viability If the cell density is in range of 1 5 2 5 x 10 cells ml and the culture is without antibiotics proceed to step 2a If the cell density is not in this range or the cell culture contains antibiotics follow steps 2b 2c 1 a Add 2 ml of Grace s Insect Medium Unsupplemented without antibiotics and serum in each well Seed 8 x 10 Sf9 or Sf21 cells from Step 1 per well Do not change medium or wash the cells The medium carried over will enhance the transfection efficiency Allow cells to attach for 15 minutes at room temperature in the hood Proceed to step 3 Prepare 10ml plating medium by mixing 1 5 ml Grace s Insect Medium Supplemented containing 10 FBS without antibiotics and 8 5 ml Grace s Insect Medium Unsupplemented without FBS and antibiotics Plate 8 x 10 Sf9 or Sf21 cells from Step 1 per well Allow cells to attach for 15
56. od claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Invitrogen Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose The buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to
57. ol Resuspend the pellet in 10 ul of sterile water Proceed to PCR Procedure below You will need to optimize PCR conditions for your specific primers and template For the PCR amplification you may use the Polyhedrin forward primer 5 AAATGATAACCATCTCGC 3 and a primer of your own design that binds within your gene of interest See page 28 for Polyhedrin forward primer binding site Continued on next page Analyzing Recombinant Viral DNA continued Analyzing PCR Results Recombination Region for BaculoDirect N GST Linear DNA Calculate the expected size of your PCR fragment based on the location of the primer binding sites see below for a diagram After running your PCR reactions on a 1 agarose gel you should see a band of the expected size for recombinant viral DNA and no bands for the negative control Features of the Recombination Region The recombination region of the recombinant baculovirus resulting from BaculoDirect N GST Linear DNA x entry clone is shown below Shaded regions correspond to DNA sequence transferred from the entry clone into the BaculoDirect N GST Linear DNA by recombination Non shaded regions are derived from the BaculoDirect N Term Linear DNA The underlined nucleotides flanking the shaded region correspond to bases 5272 and 10716 respectively of the BaculoDirect N GST Linear DNA sequence Polyhedrin Forward priming site 4397 TGGAAATGTC TATCAATATA TAGTTGC
58. or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is n
59. ot willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany This recombinant baculovirus expression system is the subject of one ore more of US patents 4 745 051 4 879 236 5 155 037 and 5 278 050 and corresponding foreign applications licensed to Invitrogen Corporation and sold for research purposes only Utilization of this product or system for the expression of gene products for commercial product development manufacturing or sale requires a license under the rights of The Texas A amp M University System Please contact Technology Licensing Manager Agriculture and Life Sciences Technology Licensing Office The Texas A amp M University System 310 Wisenbaker College Station TX 77843 3369 Phone 409 847 8682 Fax 409 845 1402 You may not distri
60. ou plan to plaque purify your virus as neutral red is a known mutagen that can alter your recombinant virus 1 Prepare al mg ml Neutral Red solution in complete growth medium and filter sterilize 2 Combine the following reagents in a 50 ml tube and place in a 37 C water bath Neutral Red 1 mg ml 1 5 ml Complete growth medium 16 5 ml 3 Microwave 4 Agarose Gel until melted then place in a 47 C water bath for 5 minutes 4 Move the 50 ml tube of neutral red solution and the 4 Agarose Gel to a sterile hood Add 6 ml of 4 Agarose Gel to the neutral red solution 5 Add 1 ml of the Neutral Red overlay to each well containing plaquing overlay Once the agarose has hardened return plates to a 27 C incubator until plaques are ready to count Plaques will appear as clear spots on a red monolayer Use the equation below to calculate your viral titer number of plaques pfu pfu ml dilution factor x ml of inoculum A well with a viral dilution of 10 contains 18 white plaques The viral titer is 18 pfu pfu ml 10 x1ml 18x 10 pfu ml Once you have a viral stock of suitable titer 2 1 x 10 pfu ml you may infect cells and perform expression studies see page 29 To plaque purify the virus or to analyze the recombinant DNA proceed to the next section Isolating Virus From a Single Plaque Introduction Materials Needed Preparing Cells Infecting Cells This section provides deta
61. pon receipt store the components as detailed All components are guaranteed for six months if stored properly Item Shipping Storage BaculoDirect GST Gateway Gel ice 4 C except Transfection Kit pENTR CAT 20 C Gateway LR Clonase II Enzyme Mix Dry ice 20 C for BaculoDirect Ganciclovir Dry ice 20 C Sf21 Frozen Cells Dry ice Liquid nitrogen Grace s Insect Cell Culture Medium Room 4 C protected from Unsupplemented Temperature light Continued on next page Kit Contents and Storage continued Transfection Kit Components Expression Kit Components vi The BaculoDirect GST Gateway Transfection Kits include the following components sufficient to perform 5 reactions Store components as detailed below Item Composition Amount Storage BaculoDirect N GST 300 ng per tube in 6 tubes 4 C Linear DNA linearized 10 ul of TE buffer pH 8 0 with Bsu36 I Cellfectin IT Reagent 1 mg ml in membrane 125 ul 4 C filtered water pENTR CAT Control 40 ul of 0 5 ng ul vectorin 20 ug 20 C Plasmid TE buffer pH 8 0 Ganciclovir 100 mM in deionized 50 ul 20 C water protected from light TE buffer pH 8 0 10 mM Tris HCl 1 mM EDTA pH 8 0 TM The BaculoDirect GST Expression Kits include the following components Store components as detailed below Item Composition Amount Storage BaculoDirect
62. ressed but also see degradation Harvesting time is not optimal Do a time course experiment and harvest cells at different time points e g 48 60 72 and 96 hrs MOI is too low e Doa plaque assay or end point dilution to accurately determine viral titer stock s Doan MOI test with different MOI e g 1 5 and 10 Continued on next page 38 Troubleshooting continued Problem Possible Cause Solution Protein is expressed but also see degradation Premature stop codon in sequence Check the sequence of your entry clone to verify that it does not contain a premature stop codon or mutations Protein is expressed but is insoluble A binding partner or other parts of protein may be needed for proper folding Identify the partner and coexpress Protein is normally secreted but is cloned without the secretion signal sequence and is now expressed intracellularly Express protein as secreted protein by adding secretion signal sequence Protein is not extracted properly For complete extraction use sonication with short pulses and Dnasel Keep samples on ice Protein is expressed but not secreted N terminal GST tag interferes with secretion Purify your protein from cell lysates Protein expressed well small scale but lost expression when scaled up MOL and harvesting time may need to fine be tuned after scaling up Using the MOI and harvest
63. sites Lambda recombination occurs between site specific attachment aft sites attB on the E coli chromosome and attP on the lambda chromosome The att sites serve as the binding site for recombination proteins and have been well characterized Weisberg amp Landy 1983 Upon lambda integration recombination occurs between attB and attP sites to give rise to attL and attR sites The actual crossover occurs between homologous 15 bp core regions on the two sites but surrounding sequences are required as they contain the binding sites for the recombination proteins Landy 1989 Continued on next page The Gateway Technology continued Gateway LR Recombination Reaction TM By using the BaculoDirect GST Gateway Transfection Kit available separately or as a part of the BaculoDirect GST Expression Kit you will take advantage of the LR reaction to transfer your gene of interest into the BaculoDirect N GST Linear DNA The LR reaction facilitates recombination of an attL substrate entry clone with an aitR substrate BaculoDirect N GST Linear DNA to create an attB containing expression virus see diagram below This reaction is catalyzed by LR Clonase II Enzyme Mix for BaculoDirect attL attL attR attR BaculoDirect N GST Linear DNA LR Clonase Il or a BaculoDirect attB attB attP attP by product expression virus Ganciclovir Introduction Ganciclovir
64. ssages e If using suspension culture for amplification cell density should be between 8 x 10 and 1 x 10 cells ml Enlarged cell diameter observed 24 hours post infection MOL is higher than 1 High MOI will decrease the viral stock quality Transfect 70 confluent cells on a six well plate with a dilution series of P1 viral stock and monitor the cells every 24 hours Use the MOI that does not produce any morphological changes within 24 hours in 70 80 of the infected cells 35 Continued on next page Troubleshooting continued Plaque Assay The following table lists some potential problems and possible solutions to help you troubleshoot your plaque assay Problem Possible Cause Solution No plaques Kinetics of infection are slower Observe plates until 8 or 9 days after than expected infection If no plaques appear investigate other possible causes No confluent monolayer on Seed 8 x 10 cells in a six well plate with Day 2 or Day 3 post infection 70 confluence Cells should double at least once before infection stops growth Excessive condensation during Remove paper towels or open the incubation at 27 C container containing plates as soon as condensation appears Viral titer too low Use higher concentrations of viral titer You may need to re infect your cells and collect a higher titer of your viral stock Small plaques that are Too many cells seeded Seed fewer cells
65. sssnenenensessnsnsesenennnnnnnnnnnennnenenennnnnnnnennnennnnnnanennnn 33 Troubleshooting aren 2m ee aa E E CERT SEES EE AER SITUS TLS SC reiten a e EEE TE 34 PADD GUA TT 40 Recipes a Sat estes a tc By ees do bans oR oven BORN 40 Map ot BaculoDirect N GST Linear DNA ea een 42 Features of the BaculoDirect N GST Linear DNA eee 43 M ap of PENIR CN Teenie are 44 Technical Supporte enei aeaea a e a SE Ae AERE essen 45 P rchaser Notification singen e e SEHR R E a TE E a E 46 References n E a ee ETT H E LIES Tove 49 iii iv Kit Contents and Storage Types of Kits This manual is supplied with the following kits Product Cat no BaculoDirect GST Gateway Transfection Kit A10640 BaculoDirect GST Gateway Expression Kit A10641 Kit Components Each kit contains the components listed below See the next page for a detailed description of other reagents supplied with each kit Component yi no Cat no 0640 A10641 BaculoDirect N GST Linear DNA v v Cellfectin II Reagent v v pENTR CAT Control Plasmid v v Ganciclovir v v Gateway LR Clonase II Enzyme Mix for BaculoDirect E Sf21 Frozen Cells v Grace s Insect Cell Culture Medium Unsupplemented v BaculoDirect GST Gateway Expression Kit Manual v v Insect Cell Lines Manual v Shipping Storage The BaculoDirect GST Gateway Transfection and Expression Kits are shipped as described below U
66. te growth medium see Note below e Sf 900 Medium 1 3X or other appropriate plaquing medium see Note below s 4 Agarose Gel e Sterile cell culture grade distilled water e 100 ml sterile glass bottle e Serial dilutions of viral stock see page 21 e Bluo gal 50 mg ml see page 41 for a recipe e Six well tissue culture plates e Sterile hood e Water baths at 47 C and 70 C e 27 C incubator e Inverted microscope e Air tight bags or containers Continued on next page 20 Performing a Plaque Assay continued Note Diluting Virus Infecting Cells with Virus 21 If you are culturing your Sf9 or Sf21 cells in serum supplemented media i e complete TNM FH you should have the following reagents on hand see page vii for ordering information e Grace s Insect Cell Culture Medium Supplemented s Grace s Insect Cell Culture Medium 2X e Fetal Bovine Serum FBS Qualified Heat Inactivated You will be infecting cells with serial dilutions of your viral stock Keep in mind the following points when preparing the 10 fold serial dilutions e Prepare dilutions in complete growth medium e Vortex viral stocks or dilutions before making the next dilution to ensure virus is evenly resuspended e Prepare 3 ml for duplicate wells or 4 ml for triplicate wells of each viral dilution e Make sure to return your viral stock to 4 C e Prepare dilutions according to the viral stock you are using to
67. the titer of the P2 viral stock see next page e Use the equation provided below to determine the amount of P2 viral stock to use to infect at a specific MOI e Scale up the amount of cells and volume of virus appropriately and follow the guidelines outlined in this section Note that ganciclovir selection is not required for generation of the P3 viral stock To amplify your viral stock infect cells at a multiplicity of infection MOI ranging from 0 1 to 1 0 MOI is defined as the number of virus particles per cell Use the following formula to calculate how much viral stock to add to obtain a specific MOI _ MOI pfu cell x number of cells viral titer pfu ml Inoculum required ml Note If you have not determined the titer of your P2 viral stock you may assume that the titer ranges from 5 x 10 to 1 x 10 pfu ml If you start with a frozen viral master stock we recommend generating a new high titer stock as viral titer generally decreases from storage at 80 C To generate another high titer stock from the master stock re infect insect cells and amplify the viral stock using the guidelines outlined in this section Now that you have a high titer viral stock you will need to determine the titer of your viral inoculum Proceed to the next section to perform a plaque assay and calculate viral titer Performing a Plaque Assay Introduction BaculoTiter Assay Kit Blue White Screening Materials Ne
68. ting Technical Support page 45 Experimental Overview Experimental The following diagram summarizes the general steps required to express your Summary gene of interest GOI using the BaculoDirect GST Gateway Expression Kit Generate a Gateway entry clone containing gt your gene of interest Entry Clone Bac loDirect NGST Perform an LR reaction between the entry clone and the BaculoDirect N GST Linear DNA to generate recombinant baculovirus DNA Directly transfect insect cells with recombinant baculovirus DNA and select with ganciclovir Collect P1 viral stock Screen for recombinant protein expression optional Infect insect cells with P1 viral stock to amplify virus and select with ganciclovir Collect P2 viral stock Screen for recombinant protein lt expression required P2 Viral Stock Infect insect cells with P2 viral stock to amplify and scale up virus Collect P3 viral stock P3 Viral Stock Titer viral stock and infect insect cells Assay for expression of recombinant fusion protein Protein Expression Continued on next page 6 Experimental Overview continued Experimental Steps The experimental steps necessary to express your protein of interest using the BaculoDirect GST Gateway Transfection and Expression Kits are outlined below For more details on each step refer to the indicated pages
69. to catalyze the LR recombination reaction The LR Clonase II Enzyme Mix for BaculoDirect combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer Use the protocol provided on the next page to perform the LR recombination reaction using LR Clonase II TM Enzyme Mix for BaculoDirect Note For the LR recombination reaction use LR Clonase II Enzyme Mix for TM TM TM BaculoDirect only do not use LR Clonase II or LR Clonase from other kits e Purified plasmid DNA of your entry clone 50 150 ng ul in TE buffer pH 8 0 Note Use PureLink HiPure Plasmid Prep Kit not a silica based miniprep kit for the purification of the entry clone e BaculoDirect N GST Linear DNA 300 ng tube provided with the BaculoDirect GST Gateway Expression and Transfection Kits e pENTR CAT control plasmid optional 100 ng ul provided with the kits s LRClonase II Enzyme Mix for BaculoDirect 20 C until immediately before use e 1X TE Buffer pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA e 25 C water bath provided with the kits keep at Continued on next page Performing the LR Recombination Reaction continued LR Recombination Perform Steps 1 4 in a sterile laminar flow hood to reduce the chances of Reaction Protocol contamination 1 To set up your sample and positive control reaction add the following components directly to the BaculoDirect N GST Linear DNA tubes containing 1
70. top codon in construct e Refer to the diagram on page 28 to verify the correct reading frame of the resulting recombinant baculovirus DNA following the LR reaction e Analyze recombinant viral DNA by PCR to confirm correct size and orientation page 10 e Sequence PCR product to verify proper reading frame for expression of the GST tag Incorrect MOI used e Run initial expression studies with an MOI of 5 and 10 e Recalculate the amount of viral stock needed to infect cells using the equation on page 19 e You may need to test a range of MOIs depending on the kinetics of expression of your recombinant protein Cells harvested too late Do a time course experiment and harvest cells at different time points e g 48 60 72 and 96 hrs Protein is lost during cell lysis If you are trying to detect an intracellular protein analyze the supernatant to determine if the protein is being lost due to cell lysis Protein is degraded or unstable e Add protease inhibitors to your cell lysates e Check mRNA levels Protein is toxic to cells Harvest cells at earlier time points e g 18 24 hours post infection 37 Continued on next page Troubleshooting continued Problem Possible Cause Solution Very little or no recombinant fusion protein detected but cells are infected and dead Viral stock a mixture of recombinant and non recombinant virus Add 100
71. ty chromatography on sulfur linked glutathione agarose Allows detection of the recombinant fusion protein with anti GST antibodies Helps solubilize the recombinant fusion protein attR1 and attR2 sites Allows recombination cloning of the gene of interest from an entry clone Immediate early promoter PIE 1 0 Allows expression of the herpes simplex virus thymidine kinase gene Kovacs et al 1991 Herpes simplex virus thymidine kinase gene HSV1 tk Allows negative selection of non recombinant virus in the presence of ganciclovir Godeau et al 1992 p10 promoter Allows expression of the lacZ gene lacZ gene Allows detection non recombinant virus through blue white screening Map of pENTR CAT Description Map pENTR CAT 3231 bp is a control vector containing the chloramphenicol acetyltransferase CAT gene The CAT gene was amplified using PCR primers containing attB recombination sites The amplified PCR product was then used in a BP recombination reaction with pDONR 221 to generate the entry clone For more information about the BP recombination reaction refer to the Gateway Technology TM with Clonase II manual part no 25 0749 Following an LR recombination reaction between pENTR CAT control vector and BaculoDirect Linear DNA CAT will be expressed as a fusion to the N terminal GST tag The molecular weight of the CAT fusion protein is approximatel
72. uM ganciclovir at the end of transfection for P1 viral production Use the same amount of ganciclovir during P2 viral amplification to select against non recombinant virus Cell density too low For protein expression using suspension culture Sf9 and Sf21 cell density should be between 2 5 x 10 and 3 0 x 10 cells ml High passage viral stock is used for protein expression Cells used for protein expression should have less than 4 passages Protein is expressed but escaped detection e If expressing a secretion protein make sure to check cells for the presence of the protein because secretion will never be 100 efficient and sometimes could be very low s If your protein of interest is expressed intracellularly make sure the check the cell lysate pellet for its presence Very little or no recombinant fusion protein detected but cells are healthy and not dying after 72 hours Viral stock is revived from frozen aliquots MOLT is too low The titer of the frozen viral stock will decrease after reviving If the titer is too low amplification may be needed e Doa plaque assay or end point dilution to accurately determine viral titer stock e Doan MOI test with different MOI e g 1 5 and 10 Cell density too high or cells are too old For protein expression using suspension culture Sf9 and Sf21 cell density should be between 2 5 x 10 and 3 0 x 10 cells ml Protein is exp
73. us Virology 202 586 605 Bushman W Thompson J F Vargas L and Landy A 1985 Control of Directionality in Lambda Site Specific Recombination Science 230 906 911 Carrington J C and Dougherty W G 1988 A Viral Cleavage Site Cassette Identification of Amino Acid Sequences Required for Tobacco Etch Virus Polyprotein Processing Proc Natl Acad Sci USA 10 3391 3395 Coligan J E Dunn B M Ploegh H L Speicher D W and Wingfield P T 1998 Current Protocols in Protein Science Current Protocols Chanda V B Ed John Wiley and Sons Inc New York Deutscher M P ed 1990 Guide to Protein Purification Vol 182 Methods in Enzymology Edited by Abelson J N and Simon M I Academic Press San Diego CA Dougherty W G Carrington J C Cary S M and Parks T D 1988 Biochemical and Mutational Analysis of a Plant Virus Polyprotein Cleavage Site EMBO J 7 1281 1287 Godeau F Saucier C and Kourilsky P 1992 Replication Inhibition by Nucleoside Analogues of a Recombinant Autographa californica Multicapsid Nuclear Polyhedrosis Virus Harboring the Herpes Thymidine Kinase Gene Driven by the IE 1 0 Promoter A New Way to Select Recombinant Baculoviruses Nuc Acids Res 20 6239 6246 King L A and Possee R D 1992 The Baculovirus Expression System A Laboratory Guide Chapman and Hall New York NY Kovacs G K Guarino L A and Summers M D 1991 Novel Regulatory Prop
74. y 53 kDa TM The map below shows the elements of the pENTR CAT control vector The vector sequence of pENTR CAT is available at www invitrogen com or by contacting Technical Support page 45 Comments for pENTR CAT 3231 nucleotides attL1 recombination site bases 569 668 CAT ORF bases 698 1354 attL2 recombination site bases 1356 1455 Kanamycin resistance gene bases 1625 2434 pUC origin bases 2555 3228 44 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail
75. your application can be found on our website at www invitrogen com Gateway For detailed information on constructing an entry clone refer to the manual for the specific entry vector you are using Points to Consider Keep the following points in mind when constructing your entry clone to be used for BaculoDirect in the recombination reaction with the BaculoDirect N GST Linear DNA N GST Linear DNA Design your gene of interest to be in frame with the N terminal GST tag after recombination Refer to page 28 for a diagram of the recombinant baculovirus DNA Tip Keep the translation reading frame of your protein of interest in frame with the AAA AAA triplet in the attL1 site of the entry clone e Make sure your insert contains a stop codon Recommended We recommend using Sf9 or Sf21 cells to generate high titer viral stocks with the Cells BaculoDirect GST Gateway Expression Kits Note that Sf21 cells are provided with the BaculoDirect GST Gateway Expression Kits We do not recommend using High Five cells to generate viral stocks due to lower transfection efficiency Once you have generated high titer viral stocks you may use Sf9 Sf21 High Five or Mimic Sf9 cells for protein expression studies See page viii for ordering information Recommended For the highest transfection efficiency we recommend using Grace s Insect Cell Media Culture Medium Unsupplemented provided with the BaculoDirect GST
76. zing your recombinant GST fusion protein is provided below For detailed instructions consult the literature provided with each product To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels and electrophoresis apparatus are available from Invitrogen Invitrogen also carries a large selection of molecular weight protein standards and staining kits For more information refer to our website at www invitrogen com or contact Technical Support page 45 To detect expression of your recombinant fusion protein by western blot analysis you may use antibodies against the N terminal GST tag available from Invitrogen or an antibody to your protein of interest The ready to use WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods See page ix for ordering information For more information refer to our website at www invitrogen com or contact Technical Support page 45 TM If you used the control plasmid pENTR CAT to produce baculovirus expressing the CAT protein you may assay for CAT expression using your method of choice CAT will be fused to the N terminal peptide containing the GST tag allowing you to use western blot analysis with an anti GST antibody CAT Antiserum

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