Baculovirus Expression System with Gateway® Technology
Contents
1. Academic Press San Diego CA Dougherty W G Parks T D Cary S M Bazan J F and Fletterick R J 1989 Characterization of the Catalytic Residues of the Tobacco Etch Virus 49 kDa Proteinase Virology 172 302 310 Kertbundit S Greve H d Deboeck F Montagu M V and Hernalsteens J P 1991 In vivo Random B glucuronidase Gene Fusions in Arabidopsis thaliana Proc Natl Acad Sci USA 88 5212 5216 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Luckow V A Lee C S Barry G F and Olins P O 1993 Efficient Generation of Infectious Recombinant Baculoviruses by Site Specific Transposon Mediated Insertion of Foreign Genes into a Baculovirus Genome Propagated in Escherichia coli J Virol 67 4566 4579 Possee R D and Howard S C 1987 Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus Nucleic Acids Res 15 10233 10248 Smith D B Davern K M Board P G Tiu W U Garcia E G and Mitchell G F 1986 Mr 26 000 Antigen of Schistosoma japonicum Recognized by Resistant WEHI 129 Mice is a Parasite Glutathione S transferase Proc Natl Acad Sci USA 83 8703 8707 2002 2008 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 34 invitrogen Corpora2. Introduction Once you have performed the LR recombination reaction you will transform competent E coli Library Efficiency DH5a Chemically Competent E coli Box 3 are included with the Baculovirus Expression System to facilitate transformation E coli Host If you have ordered the individual Gateway pDEST vectors you will need competent F coli We recommend that you propagate vectors containing inserts in E coli strains that are recA and end A deficient such as TOP10 Cat no C4040 03 or DH5a T1 Cat no 12297 016 Avoid using an E coli strain containing an F episome The F episome contains the ccdA gene and prevents negative selection of the clone with ccdB Materials Needed LB plates containing 100 ug ml ampicillin two for each transformation warm at 37 C for 30 minutes e 42 C water bath e 37 C shaking and non shaking incubator e Library Efficiency DH5a Chemically Competent E coli see page vii or appropriate competent cells see above e S O C Medium Library Efficiency DH5a competent cells are supplied in 5 tubes containing 0 2 ml Note of competent cells each Each tube contains enough competent cells to perform 4 transformations using 50 ul of cells per transformation Once you have thawed a tube of competent cells discard any unused cells Do not re freeze cells as repeated freezing thawing will result in loss of transformation efficiency Transformation 1 For each transfo
3. LR Clonase II Enzyme Mix Box 2 Store Box 2 at 20 C for up to 6 months For long term storage keep at 80 C TE Buffer pH 8 0 Reagent Composition Amount LR Clonase II enzyme mix Proprietary 40 ul Proteinase K 2 mg ml in 40 ul 10 mM Tris HCl pH 7 5 20 mM CaCl 50 glycerol pENTR gus Positive Control 20 ul of vector at 50ng plin lpg The Library Efficiency DH5a Competent E coli kit Box 3 includes the following items Transformation efficiency is gt 1 x 10 cfu ug DNA Store Box 3 at 80 C Reagent Composition Amount Library Efficiency Chemically 5 x 200 ul Competent DH5a S O C Medium 2 tryptone 2 x6 ml may be stored at room 0 5 yeast extract temperature or 4 C 10 mM NaCl 2 5 mM KCl 10 mM MgCl 10 mM MgSO 20 mM glucose pUC19 Control DNA 10 pg ul in 5 mM Tris HCI 50 ul 0 5 mM EDTA pH 8 0 Genotype of DH5a F recAl endA1 hsdR17 rx mx supE44 X thi 1 gyrA96 relA1 vi Accessory Products Introduction Additional Products The products listed in this section are intended for use with the Baculovirus Expression System with Gateway Technology For more information refer to our website at www invitrogen com or call Technical Support see page 28 The following products are available separately from Invitrogen Product Amount Cat no Library Efficien
4. i continued on next page 11 Creating an Expression Clone continued Recombination The recombination region of the expression clone resulting from pDEST 20 x Region of entry clone is shown below The complete sequence of pDEST 20 is available for pDEST 20 downloading at www invitrogen com or from Technical Support see page 28 For a map and a description of the features of pDEST 20 refer to pages 25 26 Features of the Recombination Region e Shaded regions correspond to the DNA sequences transferred from the entry clone into pDEST 20 by recombination Non shaded regions are derived from pDEST 20 e The nucleotides flanking the shaded region correspond to bases 849 and 2532 respectively of pDEST 20 e The glutathione S transferase GST gene is indicated 65 AA start EB TACTGTTTTC GTAACAGTTT TGTAATAAAA AAACCTATAA ATATTCCGGA TTATTCATAC PpH Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln 137 CGTCCCACCA TCGGGCGCGGA TCC ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu 206 CCC ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG TAT GAG CGC GAT GAA Glutathione S transferase Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile 272 GGT GAT AAA TGG CGA AAC AAA AAG TTT GAA TTG GGT TTG GAG TTT CCC AAT CTT CCT TAT TAT ATT Asp Gly Asp Val
5. available for downloading at www invitrogen com or from Technical Support see page 28 For a map and a description of the features of pDEST 10 refer to pages 24 and 26 Features of the Recombination Region e Shaded regions correspond to the DNA sequences transferred from the entry clone into pDEST 10 by recombination Non shaded regions are derived from pDEST 10 e The nucleotides flanking the shaded region correspond to bases 344 and 2168 respectively of pDEST 10 155 ca transcription start O TTACTGTTTT CGTAACAGTT TTATTCATAA AATGACAAAA GCATTGTCAA Pon Met Ser Tyr Tyr His His His His His 235 ATCGGGCGCG GATCTCGGTC CGAAACC ATG TCG TAC TAC CAT CAC CAT CAC CAT TAGCCCGCGC CTAGAGCCAG GCTTTGG TAC AGC ATG ATG GTA GTG GTA GTG GTA TEV recognition site 344 I l Glu Asn Leu Tyr Phe Gln Gly Ile Thr Ser Leu GAA AAC CTG TAT TTT CAG GGC ATC ACA AGT TTE CTT TTG GAC ATA AAA GTCACCG TAG TGT TCA AAC TTGTAATAAA AAAACCTATA AATATTCCGG ATTATTCATA CCGTCCCACC AACATTATTT TTTTGGATAT TTATAAGGCC TAATAAGTAT GGCAGGGTGG 6x His tag Tyr Lys Lys Ala Gly TAC AAA AAA GCA GGC TNN z2 ATG TTT HHMMGGIMGEG ANN Lesse TEV cleavage site 2168 TCTTGTACAA AGTGGTGATG CCATGGATCC GGAATTCAAA AGAACATGTT TCACCACTAC GGTACCTAGG CCTTAAGTTT attB2 attB1 GGCCTACGTC GACGAGCTCA CCGGATGCAG CTGCTCGAGT His Asp Tyr Asp Ile Pro Thr Thr CAC GAT TAC GAT ATC CCA ACG ACC GTG CTA ATG CTA TAG GGT TGC TGG NACCCAGCTT NTGGGTCGAA
6. bases 5013 5293 Tn7 right arm bases 5701 5924 Gentamicin resistance ORF bases 5991 6524 C Prokaryotic promoter bases 6713 6740 C C Complementary strand 25 Features of pDEST 8 pDEST 10 and pDEST 20 Features 26 The features of pDEST 8 6526 bp pDEST 10 6708 bp and pDEST 20 7066 bp are described below All features have been functionally tested Features Function Polyhedrin promoter Allows efficient high level expression of your recombinant protein Possee and Howard 1987 Mini Tn7 element Tn7R and Tn7L Allows site specific transposition of your gene of interest into a bacmid propagated in E coli Craig 1989 Luckow et al 1993 N terminal 6xHis tag in pDEST 10 only Permits purification of your recombinant protein on metal chelating resins such as ProBond N terminal glutathione S transferase GST tag in pDEST 20 only Allows affinity purification of recombinant fusion protein using glutathione agarose TEV cleavage site in pDEST 10 only Allows removal of the N terminal polyhistidine tag from your recombinant protein using AcTEV protease Carrington and Dougherty 1988 attR1 and attR2 sites Bacteriophage A derived DNA recombination sequences that allow recombinational cloning of the gene of interest from a Gateway entry clone Landy 1989 Chloramphenicol resistance gene Permits counterselection of the ex
7. the transfected cells and used to infect fresh insect cells for protein expression purification and analysis see diagram below The figure below depicts the generation of recombinant baculovirus and the expression of your gene of interest using the Bac to Bac Baculovirus Expression System en er Helper p p AN Transposition r Ar ud enced Ar ae pPoln Competent DH10Bac E coli Cells E coli LacZ Containing Recombinant Bacmid Mini prep of High molecular Weight DNA Pennen or Determine Viral Titer by Plaque Assay 1 Transfect Insect Cells with Cellfectin Reagent O00 000000 0 Saas Expression Clone Clone Recombinant Baculovirus Particles Infect Insect Cells 0000000000 Y Recombinant Gene Expression or Viral Amplification Recombinant Bacmid DNA Experimental Overview Experimental The figure below describes the steps necessary to clone and express your gene of Outline interest using pDEST 8 pDEST 10 or pDEST 20 pDEST Vector Entry Clone y Gateway Expression Clone Transform into MAX Efficiency DH10Bac Cells containing bacmid and helper y E coli Colonies with Recombinant Bacmid Restreak Optional Y Verified E coli Colonies with Recombinant Bacmid Isol
8. you can affinity purify your protein using glutathione agarose Refer to the Agarose manufacturer s instructions to purify your protein Using AcTEV AcTEV Protease is a site specific protease recognizing the seven amino acid Protease sequence Glu As Le Tyr Ph Gln Gly The cleavage site is between Gln and Gly Dougherty et al 1989 Recombinant AcTEV Protease is available from Invitrogen see page vii Use the AcTEV Protease to cleave the 6xHis tag from the fusion protein generated using pDEST 10 after purifying the recombinant protein on a nickel chelating resin The Recombinant AcTEV Protease is engineered with a 6xHis tag to facilitate removal of the enzyme from the protein sample after digestion Note After TEV cleavage at least 10 amino acids will remain at the N terminus of your protein see diagram on page 11 TM For detailed protocols refer to the ACTEV Protease manual available at www invitrogen com or by contacting Technical Support see page 28 22 Appendix Map of pDEST 8 pDEST 8 Map TM The figure below summarizes the features of the pDEST 8 vector 6526 bp For a more detailed explanation of each feature see page 26 The sequence of pDEST 8 is available from our website www invitrogen com or from Technical Support see page 28 This vector has not been completely sequenced It was compiled from published sequence data and actual sequence data If you suspect an error c
9. CAA GGC GCA GTA TTA GTT TGT TCA AAC ATG TTT mms L 2532 atte I TNN GENE NACCCAGCTT TCTTGTACAA AGTGGTTTGA TAGCTTGTCG AGAAGTACTA GAGGATCATA END TT NTGGGTCGAA AGAACATGTT TCACCAAACT ATCGAACAGC TCTTCATGAT CTCCTAGTAT l attB2 12 Performing the LR Recombination Reaction Introduction Once you have produced an entry clone containing your gene of interest you are ready to perform an LR recombination reaction between the entry clone and the appropriate pDEST vector and to transform the reaction mixture into Library Efficiency DH5a to select for an expression clone It is important to have everything you need set up and ready to use to ensure that you obtain the best results We suggest that you read this section and the one entitled Transforming Library Efficiency DH5a Cells page 15 before beginning We also recommend that you include a positive control see below and a negative control no LR Clonase II in your experiment Positive Control The pENTR gus plasmid is included in the Baculovirus Expression System with Gateway Technology for use as a positive control for LR recombination and expression Use of the pENTR gus entry clone in an LR recombination reaction with any pDEST vector will allow you to generate an expression clone containing the gene encoding B glucuronidase gus LR Clonase Il LR Clonase II enzyme mix is supplied with the kit Cat no 11827 011 only or Enzyme Mix availabl
10. Invitrogen Baculovirus Expression System with Gateway Technology Gateway adapted destination vectors for cloning and high level expression of recombinant proteins in Baculovirus Catalog nos 11827 011 11806 015 11804 010 11807 013 Version F 1July 2008 25 0516 ii Table of Contents Table gf Contents is seas eae ette ete etit ATAUT iii Kit Gontents and Storage isi antep pete en eee c died d E e ie et ie E ed ri niger v Accessory PrOQdUCtS cimo nete dem anderson Cra di de dans e AA tes vii nnn 1 OVERVIEW neen usn ee io S e n WDE a er ttes DEL e d AR I EC a BERE dee dou 1 Bac to Bac Baculovirus Expression Syste iso sepius ee EE va m ap ap aa este de 3 Experimental Overview onsen tekeer sinned ans nig atl n ard nte pete uper tra Re i 5 MethodsS E 7 Culturing Insect Cells eret ette me Fd eee tee tene Fe See Sea nese eene 7 Generating an Entry Clone iiie SUR OE nea tea e p ia dee Ur eene venete eter a 8 Creating an Expression Clone teneret ide trie hl ben stereo nha faeetet he ieu lon ip esee torba fane kokana ERSS 9 Performing the LR Recombination Reaction nnn anenenenenenenenenenseersenenenevenseneneneneneneenenenenenenenenens 13 Transforming Library Efficiency DH5a Cells onus aea RHEIN duane 15 Analyzing Transformants ve esee e tede nee ede eed RP p ODE SSD en 16 Expressing Your Protein Using the Bac to Bac Baculovirus Expression System nennen sss 18 Testing for Expression a
11. Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met 338 GAT GGT GAT GTT AAA TTA ACA CAG TCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC ATG Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg 404 TTG GGT GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA ATG CTT GAA GGA GCG GTT TTG GAT ATT AGA Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys 470 TAC GGT GTT TCG AGA ATT GCA TAT AGT AAA GAC TTT GAA ACT CTC AAA GTT GAT TTT CTT AGC AAG Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His 536 CTA CCT GAA ATG CTG AAA ATG TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT GAT CAT Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys 602 GTA ACC CAT CCT GAC TTC ATG TTG TAT GAC GCT CTT GAT GTT GTT TTA TAC ATG GAC CCA ATG TGC Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys 668 CTG GAT GCG TTC CCA AAA TTA GTT TGT TTT AAA AAA CGT ATT GAA GCT ATC CCA CAA ATT GAT AAG Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly 734 TAC TTG AAA TCC AGC AAG TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC ACG TTT GGT GGT GGC 849 l Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg His Asn Gln Thr Ser Leu Tyr Lys Lys Ala Gly 800 GAC CAT CCT CCA AAA TCG GAT CTG GTT CCG CGT CAT AAT CAA ACA AGT TTG TAC AAA AAA GCA GGC CTA GAC
12. and chloramphenicol sensitive Transformants containing a plasmid with a mutated ccdB gene will be both ampicillin and chloramphenicol resistant To check your putative expression clone test for growth on LB plates containing 30 ug ml chloramphenicol A true expression clone will not grow in the presence of chloramphenicol To confirm that your gene of interest is in frame with the appropriate tags you may want to sequence your expression construct continued on next page Analyzing Transformants continued Long Term Storage Once you have confirmed that you have the correct expression clone prepare a glycerol stock for long term storage We also recommend keeping a stock of plasmid DNA at 20 C To prepare a glycerol stock 1 Grow the E coli strain containing the plasmid overnight in selective medium Combine 0 85 ml of the overnight culture with 0 15 ml of sterile glycerol Vortex and transfer to a labeled cryovial Freeze the tube in liquid nitrogen or dry ice ethanol bath and store at 80 C 17 Expressing Your Protein Using the Bac to Bac Baculovirus Expression System Introduction Preparing Plasmid DNA Materials Supplied by the User Bac to Bac Baculovirus Expression 18 Once you have your expression clone you are ready to transform your clone into MAX Efficiency DH10Bac Chemically Competent E coli and express your protein in the desired insect cell line using the Bac to Ba
13. ate recombinant bacmid DNA Y Recombinant Bacmid DNA Transfect insect cells with Cellfectin II Reagent y P1 Recombinant Baculovirus Stock gt 10 pfu ml Infect insect cells to amplify virus P2 Recombinant Baculovirus Stock gt 107 pfu ml Titer and infect insect cells Protein Expression continued on next page Experimental Overview continued Materials Needed e Entry clone containing your gene of interest see page 8 e Insect cell lines see page 7 e Media for insect cells e Cellfectin II Reagent e Appropriate tissue culture plates and flasks e Sterile microcentrifuge tubes 1 5 ml e MAX Efficiency DH10Bac Chemically Competent E coli see page vii for ordering information If you have ordered the individual Gateway pDEST vectors you will also need TM e LRClonase II enzyme mix see page vii e Library Efficiency DH5a Chemically Competent Cells or appropriate competent cells see page vii Methods Culturing Insect Cells Introduction Cells for Transfection Insect Cell Lines Manual Before you start your cloning experiments be sure to have cultures of Sf9 Sf21 or High Five cells growing and have frozen master stocks available You will need log phase cells with gt 95 viability to perform a successful transfection Refer to the Bac to Bac Baculovirus Expression System manual to determine h
14. between your entry clone and the destination vector If you want to include a negative control Reaction 14 TM set up a separate reaction but omit the LR Clonase IT enzyme mix 1 Add the following reagents to 1 5 ml microcentrifuge tubes at room temperature and mix Component Sample Positive Control Entry clone 50 150 ng reaction 1 7 ul Destination vector 150 ng ul 1 ul 1 ul pENTR gus 50 ng ul 2 ul 1X TE Buffer pH 8 0 to 8 ul 5 pl De TM Remove the LR Clonase IT enzyme mix from 20 C and thaw on ice 2 minutes Vortex the LR Clonase II enzyme mix briefly twice 2 seconds each time TM To each sample add 2 ul of LR Clonase II enzyme mix Mix well by pipetting up and down TM Reminder Immediately return the LR Clonase II enzyme mix to 20 C Incubate reactions at 25 C for 1 hour Note For most applications 1 hour will yield a sufficient number of colonies for analysis Depending on your needs the length of the recombination reaction can be extended up to 18 hours For large plasmids 10 kb longer incubation will yield more colonies Add 1 ul of Proteinase K solution to each reaction Incubate for 10 minutes at 37 C Proceed to Transforming Library Efficiency DH5a TM Cells next page Note You may store the LR reaction at 20 C for up to 1 week before transformation if desired Transforming Library Efficiency DH5a Cells
15. c Baculovirus Expression System Prepare plasmid DNA from the selected expression clone for transformation We recommend isolating plasmid DNA using the S N A P MiniPrep Kit Cat no K1900 01 PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 or CsCl gradient centrifugation You will need the following items before starting e Insect cell line see page 7 e Appropriate cell culture media e Cellfectin II Reagent see page vii for ordering information e MAX Efficiency DH10Bac Chemically Competent E coli see page vii for ordering information Refer to the Bac to Bac Baculovirus Expression System manual for detailed protocols to perform the steps outlined below For more information on the Bac to Bac Baculovirus Expression System and insect cell culture techniques refer to the Guide to Baculovirus Expression Vector Systems and Insect Cell Culture Techniques These manuals are available from our website at www invitrogen com or by contacting Technical Support see page 28 You will need to perform the following steps to express your protein of interest from the expression clone using the Bac to Bac Baculovirus Expression System 1 Transform plasmid DNA from the expression clone into MAX Efficiency DH10Bac Chemically Competent E coli 2 Isolate recombinant bacmid DNA Verify transposition to the bacmid using PCR analysis 3 Transfect the desired insect cell line with the reco
16. ce gene bases 2990 3805 C C complementary strand 27 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 techsupport invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds Certificate of The Certificate of Analysis provides detailed quality control information for Analysis each product Certificates of Analysis are available on our website Go to www inv
17. 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 distribute 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 This product is the subject of WIPO patent WO8809372 and foreign equivalents to be used for scientific investigation and research and for no other purpose whatsoever Licenses for commercial use of the above mentioned patents must be negotiated directly with Amrad Corporation 576 Swan Street Richmond Victoria Australia 3121 Telephone 61 3 9208 4000 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 ope
18. cy DH5a Chemically 5 x 0 2 ml 18263 012 Competent Cells LR Clonase IT Enzyme Mix 20 reactions 11791 020 100 reactions 11791 100 Cellfectin II Reagent 1 ml 10362 100 MAX Efficiency DH10Bac Competent E coli 5 x 100 ul 10361 012 Express Five SFM 1000 ml 10486 025 Sf 900 II SFM 1X liquid 500 ml 10902 096 High Five Frozen Cells 3 x 10 cells ml B855 02 Sf9 Frozen Cells 1 ml 10 cells ml B825 01 Sf21 Frozen Cells 1 ml 10 cells ml B821 01 ProBond Purification System 6 purifications K850 01 ProBond Nickel Chelating Resin 50 ml R801 01 Purification Columns 50 columns R640 50 10 ml polypropylene columns Ni NTA Purification System 6 purifications K950 01 AcTEV Protease 1 000 units 12575 015 10 000 units 12575 023 vii viii Overview Introduction Features of the Destination Vectors Introduction The Baculovirus Expression System with Gateway Technology allows you to express your gene of interest in insect cell lines using the Bac to Bac Baculovirus Expression System For more information on the Bac to Bac Baculovirus Expression System see page 3 The kit uses Gateway Technology to create an expression clone by recombining an entry clone containing your gene of interest with a destination vector pDEST of choice For more information on the Gateway Technology see the next page Depending on the vector chosen the pDEST vectors allow production of native or N terminal tagge
19. d on next page Overview continued Gateway Technology LR Recombination Reaction The Gateway Technology is a universal cloning method that takes advantage of the site specific recombination properties of bacteriophage lambda Landy 1989 to provide a rapid and highly efficient way to move your gene of interest into multiple expression systems To express your gene of interest using the Gateway Technology 1 Clone your gene of interest into a Gateway entry vector of choice to create an entry clone 2 Perform an LR recombination reaction between the entry clone and a Gateway destination vector e g pDEST 8 pDEST 10 pDEST 20 3 Transform Library Efficiency DH5a E coli and select for an expression clone Use your expression clone in the Bac to Bac Baculovirus Expression System to generate a recombinant baculovirus that expresses your recombinant protein For more detailed information about the Gateway Technology refer to the Gateway Technology with Clonase II manual This manual is supplied with the Baculovirus Expression System with Gateway Technology and is also available for downloading at www invitrogen com or by contacting Technical Support see page 28 You will perform an LR recombination reaction between the entry clone and your destination vector of choice to generate an expression clone The LR recombination reaction is mediated by LR Clonase II Enzyme Mix a mixture of the bacteriophage 4 In
20. d recombinant proteins see table below Vector Fusion Peptide Fusion Tag pDEST 8 pDEST 10 N terminal 6xHis pDEST 20 N terminal Glutathione S transferase GST Smith et al 1986 pDEST 8 pDEST 10 and pDEST 20 have the following features e The polyhedrin gene promoter from Autographa californica multi nuclear polyhedrosis virus AcMNPV for high level expression of the gene of interest Possee and Howard 1987 e Mini Tn7 elements for site specific transposition into the bacmid DNA propagated in E coli Craig 1989 Luckow et al 1993 e N terminal fusion tags for detection and purification of recombinant fusion proteins choice of tag depends on the particular vector see above e Two recombination sites attR1 and attR2 for recombinational cloning of the gene of interest from an entry clone e Chloramphenicol resistance gene located between the two attR sites for counterselection e The ccdB gene located between the two attR sites for negative selection e The SV40 polyadenylation signal for efficient transcription termination and polyadenylation of mRNA e Ampicillin resistance gene for selection of transformants in E coli e Gentamicin resistance gene for selection of transformants containing recombinant bacmid DNA e The pUC origin for high copy replication and maintenance of the plasmid in E coli For more information and maps of these vectors see pages 23 26 continue
21. dB gene bases 1712 2017 attR2 recombination site bases 2058 2182 SV40 late polyadenylation signal bases 2304 2540 Tn7 left arm bases 2563 2757 F1 intergenic region bases 2922 3377 bla promoter bases 3410 3514 Ampicillin resistance ORF bla bases 3509 4369 pUC origin bases 4510 5164 Tn7 right arm bases 5432 5655 Gentamicin resistance ORF bases 5722 6255 C Prokaryotic promoter bases 6444 6471 C C Complementary strand Map of pDEST 20 pDEST 20 Map TM The figure below summarizes the features of the pDEST 20 vector 7066 bp For a more detailed explanation of each feature see the next page The sequence of pDEST 20 is available from our website www invitrogen com or from Technical Support see page 28 This vector has not been completely sequenced It was compiled from published sequence data and actual sequence data If you suspect an error contact Technical Support see page 28 KESA atiR1 CmR ccdB attR2 Features of pDEST 20 7066 nucleotides AcMNPV polyhedrin promoter bases 46 153 GST ORF bases 161 832 attR1 recombination site bases 842 966 Chloramphenicol resistance gene bases 1075 1734 ccdB gene bases 2076 2381 attR2 recombination site bases 2422 2546 SV40 late polyadenylation signal bases 2636 2814 Tn7 left arm bases 2846 3027 f1 intergenic region bases 3191 3646 bla promoter bases 3679 3783 Ampicillin resistance ORF bla bases 3778 4638 pUC origin
22. duct 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 distribute such materials Transfer of such materials and or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications 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 the above patents based upon the manufacture use or
23. e separately from Invitrogen to catalyze the LR recombination reaction The LR Clonase II enzyme mix combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer previously supplied as separate components in LR Clonase enzyme mix into an optimized single tube format for easier set up of the LR recombination reaction Use the protocol provided on page 14 to perform the LR recombination reaction using LR Clonase II enzyme mix Note You may perform the LR recombination reaction using LR Clonase enzyme mix if desired To use LR Clonase enzyme mix follow the protocol provided with the product Do not use the protocol for LR Clonase II enzyme mix provided in this manual as reaction conditions differ Materials Needed e Entry clone containing your gene of interest 50 150 ng ul in TE pH 8 0 e pDEST vector 150 ng ul in TE pH 8 0 e pENTR gus positive control if desired supplied with the LR Clonase II enzyme mix Box 2 50 ng ul in TE pH 8 0 e LRClonase II enzyme mix Box 2 keep at 20 C until immediately before use e TE Buffer pH 8 0 10 mM Tris HCI pH 8 0 1 mM EDTA e Proteinase K solution supplied with the LR Clonase II enzyme mix thaw and keep on ice until use e Water bath set at 37 C e 1 5 ml microcentrifuge tubes continued on next page 13 Performing the LR Recombination Reaction continued LR Recombination Follow this procedure to perform the LR recombination reaction
24. echnical Support see page 28 It is important to have a properly designed entry clone before recombining with the destination vector Refer to the table below and the recombination region on pages 10 12 If you wish to Then recombine your entry clone your insert should contain an ATG start codon with pDEST 8 for proper initiation of translation and a stop codon include the 6xHis tag the entry clone must be designed to ensure that pDEST 10 your gene of interest is in frame with the ATG and the 6xHis tag after recombination and must contain a stop codon include the GST fusion tag the entry clone must be designed to ensure that pDEST 20 your gene of interest is in frame with the ATG and the GST tag after recombination and must contain a stop codon Creating an Expression Clone Introduction Experimental Outline Important Propagating the Vectors After you have generated an entry clone you will perform the LR recombination reaction to transfer the gene of interest into the pDEST vector to create your expression clone To ensure that you obtain the best possible results we recommend that you read this section and the next section entitled Performing the LR Recombination Reaction pages 13 14 before beginning To generate an expression clone you will 1 Perform an LR recombination reaction using the attL containing entry clone and the attR containing pDEST vecto
25. ency DH5a Competent E coli Shipping Storage TM II Enzyme Mix lt lt lt 2 4 The Baculovirus Expression System with Gateway Technology is shipped as described below Upon receipt store each box as detailed below Box Component Shipping Storage pDEST Vectors Wet ice 20 C Gateway LR Clonase II Enzyme Mix Dry ice 20 C Library Efficiency DH5a Chemically Dry ice 80 C Competent E coli Kit Note The individual Gateway pDEST vectors Cat nos 11804 010 11806 015 11807 013 are shipped on wet ice Upon receipt store at 20 C continued on next page Kit Contents and Storage continued Destination Vectors LR Clonase II Enzyme Mix DH5a Competent E coli The following destination vectors Box 1 are supplied with the Baculovirus Expression System with Gateway Technology Store the vectors at 20 C Note Catalog nos 11804 010 11806 015 11807 013 contain 40 ul of 150 ng ul of the appropriate pDEST vector in 10 mM TrisHCI 1 mM EDTA pH 8 0 only Reagent Composition Amount pDEST 8 Vector 40 ul of vector at 150 ng ul in TE Buffer pH 8 0 10 mM TrisHCI 1 mM EDTA pH 8 0 6 ug pDEST 10 Vector 40 ul of vector at 150 ng ul in TE Buffer pH 8 0 6 ug pDEST 20 Vector 40 ul of vector at 150 ng ul in TE Buffer pH 8 0 6 ng The following reagents are supplied with the Gateway
26. ense No 69 Baculovirus Vectors and Reagents Limited Use Label License No 125 GST 32 This product is the subject of U S Patent No 5 348 886 This product is sold under patent license from Monsanto for research purposes only and no license for commercial use is included Requests for licenses for commercial manufacture or use should be directed to Director Monsanto Corporate Research 800 N Lindbergh St Louis Missouri 63167 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 The GUS positive control vector in these products is claimed in patents and patent applications See U S Patent No 5 599 670 and Great Britain Patent No 2 197 653 licensed to Invitrogen by Cambia Biosystems L L C CBL Use of the GUS gene is restricted to use as a positive control Any other use may require a license from CBL 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
27. eway 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 33 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 New York Greene Publishing Associates and Wiley Interscience Barry G F 1988 A Broad Host Range Shuttle System for Gene Insertion into the Chromosomes of Gram negative Bacteria Gene 71 75 84 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 Ciccarone V C Polayes D and Luckow V A 1997 Generation of Recombinant Baculovirus DNA in E coli Using Baculovirus Shuttle Vector Volume 13 U Reischt ed Totowa NJ Humana Press Inc Coligan J E Dunn B M Ploegh H L Speicher D W and Wingfield P T 1998 Current Protocols in Protein Science V B Chanda ed New York John Wiley and Sons Inc Craig N L 1989 Transposon Tn7 D E Berg and H H Howe eds Washington D C American Society for Microbiology Deutscher M P 1990 Guide to Protein Purification In Methods in Enzymology Vol 182 J N Abelson and M I Simon eds
28. ion region of the expression clone resulting from pDEST 8 x entry Region of clone is shown below The complete sequence of pDEST 8 is available for pDEST 8 downloading at www invitrogen com or from Technical Support see page 28 For a map and a description of the features of pDEST 8 refer to pages 23 and 26 Features of the Recombination Region e Shaded regions correspond to the DNA sequences transferred from the entry clone into pDEST 8 by recombination Non shaded regions are derived from pDEST 8 e The nucleotides flanking the shaded region correspond to bases 167 and 1991 respectively of pDEST 8 60 uS po transcription start la ATTTTACTGT TTTCGTAACA GTTTTGTAAT AAAAAAACCT ATAAATATTC CGGATTATTC TATTTATTCA TAAAATGACA AAAGCATTGT CAAAACATTA TTTTTTTGGA TATTTATAAG GCCTAATAAG Pou 167 130 ATACCGTCCC ACCATCGGGC GCGGATCATC ACAAGTTTGT ACAAAAAAGC AGGCTNN GENE NACCCAGCTT TATGGCAGGG TGGTAGCCCG CGCCTAGTAG TGTTCAAACA TGTTTTTTCG TCCGANN 2 NTGGGTCGAA l attBl 1991 TCTTGTACAA AGTGGTGATA GCTTGTCGAG AAGTACTAGA GGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT AGAACATGTT TOACUACTAT CGAACAGCTC TTCATGATCT CCTAGTATTA GTCGGTATGG TGTAAACATC TCCAAAATGA attB2 continued on next page 10 Creating an Expression Clone continued Recombination Region of pDEST 10 313 The recombination region of the expression clone resulting from pDEST 10 x entry clone is shown below The complete sequence of pDEST 10 is
29. itrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box continued on next page 28 Technical Support continued Limited Warranty Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen 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 ou
30. ls 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 not 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 continued on next page P
31. mbinant bacmid DNA using Cellfectin II Reagent 4 Harvest the recombinant baculovirus Remember to store the virus stocks at 4 C protected from light For long term storage store at 80 C Amplify viral stocks Titer the viral stock and infect insect cells with recombinant baculovirus particles using an optimal MOI 7 Harvest cells or media at 24 48 72 and 96 hours post infection and assay for expression see next page Testing for Expression Introduction Polyacrylamide Gel Electrophoresis Western Analysis Analyzing Expression by Recombinant Viruses B Glucuronidase Assay Guidelines are provided in this section for testing the expression of your protein and the protein expressed from the positive control vector pENTR gus To facilitate separation of your expressed protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels are available from Invitrogen In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits For more information about the appropriate gels standards and stains to use to visualize your recombinant protein refer to our website www invitrogen com or call Technical Support see page 28 To detect expression of your protein by western blot analysis you may use an antibody to your protein of interest WesternBreeze Chromogenic Kits and WesternBreeze Che
32. miluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods For more information refer to our website www invitrogen com or call Technical Support see page 28 Analysis of recombinant virus expression is performed in 24 well plates using the virus stock harvested 72 hours post infection 1 Seed 6 x 10 insect cells per well in a 24 well plate Allow the cells to attach for at least 30 minutes Wash the cells once with fresh media and replace with 300 ul of fresh media Add 200 ul of viral stock to each well Include controls that contain uninfected cells wild type infected cells Incubate the plate at 27 C for 48 hours Remove the viral supernatant and save for analysis Wash the cells with SFM and lyse cells with 400 ul of 1X SDS PAGE sample buffer 7 Boilthe samples for 3 minutes Load 20 ul of the sample on an appropriate polyacrylamide gel and perform electrophoresis An assay for analyzing B glucuronidase gus activity from the positive control vector p ENTR gus is described in the Bac to Bac Baculovirus Expression System manual to verify the recombination reaction and expression continued on next page 19 Testing for Expression continued 20 Note Expression of your protein with the N terminal tag will increase the size of your recombinant protein The table below lists increase in the molecular weight of your recombi
33. n 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 attB1 and attB2 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 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 Gat
34. n7 transposition function in trans Barry 1988 The helper plasmid is present in DH10Bac and confers resistance to tetracycline and encodes the transposase continued on next page 3 Bac to Bac Baculovirus Expression System continued pDEST Vectors Diagram of the Bac to Bac Expression System Gateway cloning gt Entry Clone X pDEST containing your gene of interest TM Each pDEST vector has a mini Tn7 containing the expression cassette Each expression cassette consists of a gentamicin resistance gene the polyhedrin promoter from AcMNPV for expression of proteins in insect cells a Gateway cloning cassette attR1 Chloramphenicol resistance gene ccdB gene and attR2 and an SV40 poly A signal inserted between the left and right arms of Tn7 The important features of the pDEST vectors are described on pages 1 and 26 The gene of interest is cloned into the Gateway cloning cassette of the pDEST vectors using an entry vector see page 8 Transposition of the mini Tn7 from the pDEST vector into the mini attTn7 attachment site on the bacmid disrupts expression of the lacZa gene resulting in white colonies of the recombinant bacmid in a background of blue colonies containing the unaltered parent bacmid The recombinant bacmid DNA is rapidly isolated from small scale cultures of the white colonies and then used to transfect insect cells Viral stocks gt 10 pfu ml are harvested from
35. nant fusion protein that you should expect from the tag in each pDEST vector Be sure to account for any additional amino acids between the fusion tag and the start of your protein Vector Fusion Tag Expected Size Increase kDa pDEST 10 N terminal 4 3 pDEST 20 N terminal 27 8 Purifying the Recombinant Protein Introduction Purifying 6xHis Tagged Recombinant Proteins Note The presence of the N terminal 6xHis tag in pDEST 10 allows purification of TM recombinant fusion protein using a nickel chelating resin such as ProBond or Ni NTA while the presence of the N terminal GST tag in pDEST 20 allows purification of recombinant fusion protein using glutathione agarose ProBond and Ni NTA resin are available separately from Invitrogen see page vii for ordering information Other metal chelating resins are suitable TM e To purify your fusion protein using ProBond or Ni NTA refer to the ProBond Purification System or Ni NTA Purification System manuals as appropriate Both manuals are available for downloading from our website www invitrogen com or by contacting Technical Support see page 28 e To purify your fusion protein using another metal chelating resin refer to the manufacturer s instructions Purifying 6xHis tagged Proteins from Medium To purify 6xHis tagged recombinant proteins from the culture medium we recommend a dialysis or ion exchange chromatography step prior t
36. ntistitem etti tet talit tertie d ser e eee pee HI a te eene 19 Purifying the Recombinant Protein sse tenete ten treten tenente tenentes 21 ADDOHODE qaot dax el bx Fl px au ex de Ea EXE FER ee Run ce errr erry 23 Map OF pDEST Bittner ien MEER ORIS oP eds a E Behe Mia rM vog da Rp 23 Map ot pDEST Oneens ba alla e bd el ERE Sogn cel fr n ad ene eenn 24 Map oEpDEST 205 20d MAST EET Mad ddl c qo dM Hooded Maii AET 25 Features of pDEST 8 pDEST I0 and pDEST 20 Moss kula sakala 26 Map Ot DENTS BUS ea iti Noni pM Feb I MU Ra CO Dn eese etre pd eed 27 Techr cal Support ood n e ee e n eee v t e te api KD e ted 28 Purchaser Notification osse eie edendo eed ud e deme tp e els 30 Gateway Clone Distribution Boeing ende Ap veto tact pa Rede e OR eee 33 Referetices ostendentes eias dedos eb fii mid 34 iii lv Kit Contents and Storage Types of Kits Kit Components This manual is supplied with the following products Kit Cat no Baculovirus Expression System with Gateway Technology 11827 011 Gateway pDEST 8 Vector 11804 010 Gateway pDEST 10 Vector 11806 015 Gateway pDEST 20 Vector 11807 013 Each product contains the following components For a detailed description of the contents of each component see the next page Component Cat no 11827 011 11804 010 11806 015 11807 013 pDEST 8 Vector pDEST 10 Vector pDEST 20 Vector Gateway LR Clonase Library Effici
37. o affinity chromatography on metal chelating resins Dialysis allows e Removal of media components that strip Ni from metal chelating resins Ion exchange chromatography allows e Removal of media components that strip Ni from metal chelating resins e Concentration of your sample for easier manipulation in subsequent purification steps Conditions for successful ion exchange chromatography will vary depending on the protein For more information refer to Current Protocols in Protein Science Coligan et al 1998 Current Protocols in Molecular Biology Unit 10 Ausubel et al 1994 or the Guide to Protein Purification Deutscher 1990 Many insect cell proteins are naturally rich in histidines with some containing stretches of six histidines When using a metal chelating resin to purify 6xHis tagged proteins these histidine rich proteins may co purify with your protein of interest The contamination can be significant if your protein is expressed at low levels We recommend that you add 5 mM imidazole to the binding buffer prior to addition of the protein mixture to the column Addition of imidazole may help to reduce background contamination by preventing proteins with low specificity from binding to the metal chelating resin continued on next page 21 Purifying the Recombinant Protein continued TM Purification Using If you express your recombinant protein as a fusion to the GST tag in pDEST 20 Glutathione
38. o generate a recombinant baculovirus using homologous recombination e Permits rapid and simultaneous isolation of multiple recombinant viruses and is suited for the expression of protein variants for structure function studies The baculovirus shuttle vector bacmid bMON14272 136 kb is used in the Bac to Bac Baculovirus Expression System The bacmid contains a low copy number mini F replicon a kanamycin resistance marker and a segment of DNA encoding the LacZa peptide from a pUC based cloning vector A short segment containing the attachment site for the bacterial transposon Tn7 mini attTn7 is inserted into the N terminus of the lacZ gene of the bacmid This insertion does not disrupt the reading frame of the LacZa peptide The bacmid propagates in E coli DH10Bac as a large plasmid that confers resistance to kanamycin and can complement a lacZ deletion present on the chromosome to form colonies that are blue Lac in the presence of a chromogenic substrate such as Bluo gal or X gal and the inducer IPTG Recombinant bacmids composite bacmids are generated by transposing a mini Tn7 element from a donor plasmid pDEST vectors to the mini attTn7 attachment site on the bacmid The Tn7 transposition functions are provided by a helper plasmid see below Refer to the diagram on the next page for a schematic representation of the Bac to Bac Baculovirus Expression System The helper plasmid pMON7124 13 2 kb provides the T
39. ontact Technical Support see page 28 attR1 NEMM ccdB ratR2 Features of pDEST 8 6526 nucleotides AcMNPV polyhedrin promoter bases 43 152 attR1 recombination site bases 160 284 Chloramphenicol resistance gene bases 534 1193 ccdB gene bases 1535 1840 attR2 recombination site bases 1881 2005 SV40 late polyadenylation signal bases 2093 2271 Tn7 left arm bases 2300 2484 f1 intergenic region bases 2648 3103 bla promoter bases 3136 3240 Ampicillin resistance ORF bla bases 3235 4095 pUC origin bases 4470 4750 Tn7 right arm bases 5157 5381 Gentamicin resistance ORF bases 5448 5981 C Prokaryotic promoter bases 6170 6197 C C Complementary strand 23 Map of pDEST 10 pDEST 10 Map The figure below summarizes the features of the pDEST 10 vector 6708 bp For 24 a more detailed explanation of each feature see page 26 The seguence of pDEST 10 is available from our website www invitrogen com or from Technical Support see page 28 This vector has not been completely sequenced It was compiled from published sequence data and actual sequence data If you suspect an error contact Technical Support see page 28 6xHis TEV attRIMCMRI ccoB fattR2 Features of pDEST 10 6708 nucleotides AcMNPV polyhedrin promoter bases 115 244 6xHis tag bases 274 291 TEV cleavage site bases 313 333 attR1 recombination site bases 337 461 Chloramphenicol resistance gene bases 711 1370 cc
40. ow many cells you will need for transfection For additional information on insect cell culture refer to the Insect Cell Lines manual and the Guide to Baculovirus Expression Vector Systems and Insect Cell Culture Techniques These manuals contain information on e Thawing frozen cells e Maintaining and passaging cells e Freezing cells e Using serum free medium e Growing cells in suspension e Scaling up cell culture These manuals are available for downloading at www invitrogen com or by contacting Technical Support see page 28 Generating an Entry Clone Introduction Cloning Considerations TM TM TM To recombine your gene of interest into pDEST 8 pDEST 10 or pDEST 20 you will need an entry clone containing the gene of interest Many entry vectors are available from Invitrogen to facilitate generation of entry clones see table below For more information about each vector see our website www invitrogen com or contact Technical Support see page 28 Vector Cat no pENTR D TOPO K2400 20 pENTR SD D TOPO K2420 20 pENTR 1A 11813 011 pENTR 2B 11816 014 pENTR 3C 11817 012 pENTR 4 11818 010 pENTR 11 11819 018 Once you have selected an entry vector refer to the manual for the specific entry vector you are using for instructions to construct an entry clone All entry vector manuals are available for downloading at www invitrogen com or by contacting T
41. pression clone ccdB gene Permits negative selection SV40 polyadenylation Efficient transcription termination and sequence polyadenylation of mRNA pUC origin Permits high copy replication and maintenance in E coli bla promoter Allows expression of the ampicillin resistance gene Ampicillin resistance gene bla Allows selection of transformants in E coli Gentamicin resistance gene Allows selection of transformants containing recombinant bacmid DNA Map of pENTR gus Description Map of Control Vector Comments for pENTR gus 3841 nucleotides pENTR gus is a 3841 bp entry clone containing the Arabidopsis thaliana gene for B glucuronidase gus Kertbundit et al 1991 The gus gene was amplified using PCR primers containing attB recombination sites The amplified PCR product was then used in a BP recombination reaction with pDONR 201 to generate the entry clone For more information about the BP recombination reaction refer to the Gateway Technology with Clonase II manual TM The figure below summarizes the features of the pENTR gus vector The complete sequence and restriction enzyme cleavage sites for pENTR gus are available from our Web www invitrogen com or by contacting Technical Support see page 28 attL1 bases 99 198 complementary strand gus gene bases 228 2039 attL2 bases 2041 2140 pUC origin bases 2200 2873 C Kanamycin resistan
42. r Note Both the entry clone and the destination vector should be supercoiled see Important Note below Transform the reaction mixture into a suitable E coli host see page 15 Select for expression clones see pages 10 12 for illustrations of the recombination region of expression clones in pDEST 8 pDEST 10 or pDEST 20 The pDEST 8 pDEST 10 and pDEST 20 vectors are supplied as supercoiled plasmids Although Invitrogen has previously recommended using a linearized destination vector for more efficient recombination further testing has found that linearization of these vectors is NOT required to obtain optimal results for any downstream application If you wish to propagate and maintain the pDEST 8 pDEST 10 or pDEST 20 vectors prior to recombination we recommend using 10 ng of the vector to transform One Shot ccdB Survival 2 T1 Chemically Competent Cells Cat no A10460 from Invitrogen The ccdB Survival 2 T1 E coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene To maintain the integrity of the vector select for transformants in media containing 50 100 ug ml ampicillin and 15 30 ug ml chloramphenicol Note Do not use general E coli cloning strains including TOP10 or DH5a for propagation and maintenance as these strains are sensitive to CcdB effects continued on next page Creating an Expression Clone continued Recombination The recombinat
43. r 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 29 Purchaser Notification Introduction Limited Use Label License No 5 Invitrogen Technology 30 Use of the Baculovirus Expression System with Gateway Technology 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 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 materia
44. rmation aliquot 50 ul of Library Efficiency DH5a Protocol Chemically Competent cells into a sterile microcentrifuge tube 2 Add 1 pl of the LR recombination reaction from Step 7 previous page into the tube containing 50 ul of Library Efficiency DH5a competent cells and mix gently Do not mix by pipetting up and down Incubate on ice for 30 minutes Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 450 ul of room temperature S O C Medium Cap the tube tightly and shake the tube horizontally 200 rpm at 37 C for 1 hour No w 8 Spread 20 ul and 100 ul from each transformation on a prewarmed selective plate and incubate overnight at 37 C We generally plate 2 different volumes to ensure that at least 1 plate has well spaced colonies An efficient LR recombination reaction should produce hundreds of colonies gt 5000 colonies if the entire LR reaction is transformed and plated 15 Analyzing Transformants Analyzing Positive 1 Pick 5 colonies from Step 8 previous page and culture them overnight in LB Clones Analyzing Transformants by PCR Confirming the Expression Clone Sequencing 16 or SOB medium containing 100 pg ml ampicillin 2 Isolate plasmid DNA using your method of choice We recommend using the S N A P MiniPrep Kit Cat no K1900 01 or the PureLink HQ Mini Plasmid Purification Kit Cat no K2100 01 available from In
45. 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 none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not 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 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 page 33 continued on next page 31 Purchaser Notification continued Limited Use Label License No 21 Bac to Bac and Bac to Bac HT Limited Use Label License No 22 Vectors amp Clones Encoding Histidine Hexamer Limited Use Label License No 23 GUS Control Vector Limited Use Label Lic
46. te 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
47. tegrase Int and Excisionase Xis proteins and the E coli Integration Host Factor IHF protein For more information about the LR recombination reaction see the Gateway Technology with Clonase II manual Bac to Bac Baculovirus Expression System Introduction Advantages of Using Site Specific Transposition Baculovirus Shuttle Vector Helper Plasmid The Bac to Bac Baculovirus Expression System is a rapid and efficient method to generate recombinant baculoviruses This method is based on site specific transposition of an expression cassette into a baculovirus shuttle vector bacmid propagated in E coli Ciccarone et al 1997 Luckow et al 1993 For more details on this system refer to the Bac to Bac Baculovirus Expression System manual and the Guide to Baculovirus Expression Vector Systems These manuals are available for downloading at www invitrogen com or by contacting Technical Support see page 28 Using site specific transposition to insert foreign genes into a bacmid propagated in E coli has the following advantages over the generation of recombinant baculoviruses in insect cells using homologous recombination e Eliminates the need for multiple rounds of plaque purification as the recombinant virus DNA isolated from selected colonies is not mixed with parental nonrecombinant virus e Requires less than 2 weeks to identify and purify a recombinant virus as compared to the 4 6 weeks required t
48. urchaser Notification continued Limited Use Label License No 19 Gateway Cloning Products Gateway Clone Distribution Policy 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 method 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 pro
49. vitrogen 3 Analyze the plasmids by restriction analysis to confirm the presence of the insert You may also analyze positive transformants using PCR For PCR primers use a primer that hybridizes within the vector and one that hybridizes within your insert You will have to determine the amplification conditions If you are using this technique for the first time you may want to perform restriction analysis in parallel Artifacts may be obtained because of mispriming or contaminating template The protocol below is provided for your convenience Other protocols are suitable Materials Needed PCR SuperMix High Fidelity Invitrogen Cat no 10790 020 Appropriate forward and reverse PCR primers 20 uM each Procedure 1 For each sample aliquot 48 ul of PCR SuperMix High Fidelity into a 0 5 ml microcentrifuge tube Add 1 ul each of the forward and reverse PCR primer 2 Pick5 colonies and resuspend them individually in 50 ul of the PCR SuperMix containing primers remember to make a patch plate to preserve the colonies for further analysis Incubate reaction for 10 minutes at 94 C to lyse cells and inactivate nucleases Amplify for 20 to 30 cycles For the final extension incubate at 72 C for 10 minutes Store at 4 C ON esp Visualize by agarose gel electrophoresis The ccdB gene mutates at a very low frequency resulting in a very low number of false positives True expression clones will be ampicillin resistant
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