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Expressway™ Lumio™ Cell-Free Expression and Detection System

1. For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com e Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches Safety Data Sheets SDSs are available at www lifetechnologies com support The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Life Technologies Corporation 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 a Life Technologies product or service contact our Technical Support Representatives All Life Technologies products are warranted to perform according to specifications stated on t
2. Add 3 ml of 10 TCA to each glass tube and incubate tubes at 4 C for 20 minutes Wet individual glass fiber GF C filters with 10 TCA and place onto the vacuum filtration device Turn the vacuum on and pour the TCA solution from each glass tube into a sample well Wash filters twice with 5 TCA Wash filters once with 100 ethanol Leave the vacuum on for 1 minute to allow the filters to dry Turn the vacuum off and remove the filters Place the filters in scintillation vials and add scintillation fluid Count samples in a scintillation counter Proceed to Calculating Protein Yield next page Continued on next page Determining Protein Yield continued Calculating Use the equations below to calculate the yield of protein obtained You will need to Protein Yield determine the pmoles of methionine present in your specific reaction Remember to account for both radiolabeled and unlabeled methionine You will also need to determine the total counts incorporated using TCA precipitation see previous page Total ids total reaction volume otal counts total cpm per 5 ul spotted x Specific activity total FONS pmoles of methionine va total reaction volume a rca precipitable counts background x pmoles methionine 5 incorporated specific activity pinoletat protein pmoles of methionine incorporated into protein number of methionines in protein Yield of protein in ug moles of protein x
3. Te Fluorescence Intensity 400 450 500 550 600 650 700 Wavelength nm Real time Lumio detection allows you to directly monitor synthesis of your Lumio tagged recombinant proteins during the Expressway in vitro synthesis reaction To perform real time detection add Lumio Green Detection Reagent directly to your synthesis reaction and incubate the reaction in a standard spectrofluorometer programmed to measure fluorescence at specified time points Note Depending on the protein of interest real time detection may have varying background We recommend that you perform in gel Lumio detection immediately following real time detection which can be done with no loss of fluorescence intensity and without the use of additional Lumio Green Detection Reagent In gel Lumio detection allows you to visualize Lumio tagged fusion proteins on a polyacrylamide gel After protein synthesis treat your Lumio tagged recombinant protein with the Lumio Green Detection Reagent and perform electrophoresis You then image the gel using a laser based scanner or an imager equipped with a UV transilluminator After Lumio in gel detection the gel can be stained with Coomassie silver or fluorescent stains for total protein content Handling the Lumio Green Detection Reagent Introduction Dermal Toxicity Evaluation Accidental Spills and Accidental Contact Disposing of the Lumio Green Reagent The Lumio Green Dete
4. Fluorescent Protein Standard vi The Lumio Green Detection Kit components are listed below Store reagents at 20 C Item Composition Amount Lumio Green Detection Reagent Proprietary 20 ul Lumio Gel Sample Buffer 4X Proprietary 5 x 200 ul Lumio In Gel Detection Enhancer Proprietary 200 ul Catalog number K9900 60 comes with the pEXP5 NT CALMLS3 control vector 10 ug for use as a positive control for protein expression The plasmid allows expression of an N terminally tagged calmodulin like 3 CALML3 fusion protein The vector is supplied as 20 ul at 0 5 pg pl in TE buffer pH 8 0 Store at 20 C Note Catalog nos K9900 70 K9900 90 V960 03 or V960 04 include an N or C terminal Lumio expression control plasmid BenchMark Fluorescent Protein Standard is supplied as seven distinct proteins 711 155 kDa in storage buffer 0 45 M Tris HCI pH 8 5 2 SDS 12 glycerol 0 0025 Coomassie G 250 Amount supplied is 125 ul To avoid freezing and thawing aliquot in small volumes and store at 20 C protected from light Additional Products Accessory Products Some of the reagents supplied in the Expressway Cell Free E coli Expression System as well as other products suitable for use with the kit are available separately from Invitrogen Ordering information is provided below For more information go to www invitrogen com or contact Technical Support page 44
5. Advantages of Lumio Technology Components of the Lumio System Tetracysteine Motif This section provides a brief overview of Lumio Technology For more information see the Lumio Green Detection Kit manual included with catalog numbers K9900 70 K9900 90 and K9900 60 and available online at www invitrogen com Using the Expressway Lumio Cell Free Expression and Detection System provides the following advantages e Lumio tagged fusion protein sensitivity at subnanogram level e Direct detection of Lumio tagged fusion proteins in the polyacrylamide gel without the need for staining or western blotting e Direct real time monitoring of the production of Lumio tagged proteins during the Expressway in vitro synthesis reaction e Detection compatible with downstream applications such as Coomassie staining silver staining fluorescent staining western blotting or mass spectrometry analysis The two major components of the Lumio System are described below e The tetracysteine Lumio tag Cys Cys Pro Gly Cys Cys When fused to a gene of interest the Lumio tag allows the expressed fusion protein from the pEXP Gateway construct to be specifically recognized by the Lumio Green Detection Reagent For more information on the tetracysteine motif see below e A biarsenical Lumio Green Detection Reagent which becomes fluorescent upon binding to recombinant proteins containing the Lumio tag Lumio
6. Product Quantity Catalog no One Shot TOP10 Chemically Competent E coli 10 reactions C4040 10 20 reactions C4040 03 Gateway LR Clonase II Enzyme Mix 20 reactions 11791 020 DNase RNase Free Distilled Water 500 ml 10977 015 RNase AWAY 250 ml 10328 011 Ampicillin 20 ml 10 mg ml 11593 019 Zeocin 1g R250 01 PureLink HQ Mini Plasmid Purification Kit 100 reactions K2100 01 PureLink PCR Purification Kit 50 reactions K3100 01 Lumio Green Detection Kit 100 in gel detections LC6090 BenchMark Protein Ladder 2x250 ul 10747 012 BenchMark Fluorescent Protein Standard 125 ul LC5928 SimplyBlue SafeStain 1L LC6060 One Shot ccdB Survival 2 T1 Chemically Competent 10 reactions A10460 Cells Products to Detect If you are expressing your Lumio tagged recombinant protein from Recombinant Fusion Protein pEXP3 DEST or pEXP4 DEST you may detect expression of your recombinant fusion protein using the Lumio Detection Reagents supplied with the kit You may also use an antibody to the appropriate epitope The table below describes the products available from Invitrogen for detection of fusion proteins expressed from these vectors The amount of antibody supplied is sufficient for 25 western blots Product Epitope Catalog no Anti HisG Antibody Detects the N R940 25 Anti HisG HRP Antibody terminal l R941 25 S f polyhistidine 6xHis Anti HisG AP Antibody tag followed by R942 25 glycine HHHHHHG v
7. modifications such as phosphorylation or glycosylation to the recombinant protein Synthesized protein requires co factors for complete activity Add required co factors to the protein synthesis reaction 34 Continued on next page Troubleshooting continued Analyzing The table below lists some potential problems and possible solutions that may Proteins help you troubleshoot your electrophoresis experiments Problem Reason Solution Multiple bands observed Proteins denatured for too long Add 1X SDS PAGE sample buffer to on the polyacrylamide gel the sample and incubate at 70 C 80 C for 10 15 minutes before loading on the gel Old S Methionine used radiolabeled samples only Use fresh S Methionine Internal ATG codons in the context of RBS like sequences e Check the sequence of your gene and search for potential RBSs with the proper spacing from internal methionines e Replace the methionine or change RBS sequence s using point mutation s Smearing on the gel Samples not precipitated with acetone Precipitate the proteins with acetone to remove background smearing Follow the protocol provided on page 24 Too much protein loaded Reduce the amount of protein loaded on the gel Gel not clean Rinse the gel briefly before exposing to film If you have stained the gel with Coomassie blue destain the gel in water or 5096 methanol 7 596 glaci
8. C during protein synthesis Add mild detergents e g up to 0 0576 Triton X 100 0 02596 sodium dodecyl maltoside 0 1 CHAPS or 0 05 Brij 58 to the reaction and Feed Buffer Add molecular chaperones to the reaction Sample not mixed before spotting on filter for TCA precipitation radiolabeled samples only Mix sample before spotting on filter for TCA precipitation Continued on next page 33 Troubleshooting continued Synthesizing Proteins continued Problem Reason Solution Control reaction produces no protein Reagents have lost activity Store reagents at 80 C Store the T7 Enzyme Mix at 20 C after initial use e Use care when freezing and thawing the Expressway E coli slyD Extract Expressway 2 5X IVPS Reaction Buffer and Expressway 2X IVPS Feed Buffer Follow handling guidelines on page 15 One or two freeze thaw cycles are acceptable Avoid multiple freeze thaw cycles RNases Reagent s contaminated with Wear gloves and use RNase free supplies when handling the reagents supplied in the kit Use RNase AWAY available from Invitrogen page vii to eliminate RNase from surfaces Protein has low biological activity Improper protein folding Reduce incubation temperature to as low as 25 C during protein synthesis required Post translational modifications The Expressway E coli slyD Extract will not introduce post translational
9. Detection System Results from sample protein synthesis reactions using pEXP3 DEST with in gel and real time Lumio detection are provided below Note that real time signal strength does not correlate to protein expression levels so performing in gel detection is recommended in addition to real time detection Figure 1 In gel detection of expressed proteins containing the Lumio sequence 1 2 3 4 5 1 pl of each protein synthesis reaction was run on a 4 12 NuPAGE gradient gel and visualized using a Typhoon laser scanner Lane 1 v crk avian sarcoma virus Lane 2 cAMP dependent protein kinase Lane 3 adenylate kinase Lane 4 creatine kinase Lane 5 No DNA control Lane M BenchMark Pre Stained Protein Ladder MEINTE Figure 2 Real Time incorporation of the Lumio tag in human ORFs 5000 v crk avian sarcoma virus 8 cAMP dependent protein kinase 4000 3000 RFU 2000 1000 0 20 40 60 80 100 120 Time Minutes 31 Troubleshooting Introduction Synthesizing Proteins Review the information in this section to troubleshoot your cell free expression experiment See the Lumio Green Detection Kit manual for troubleshooting Lumio Technology The table below lists some potential problems and possible solutions that may help you troubleshoot your protein synthesis experiments Problem Reason Solution but c
10. What You Should See Confirming the Expression Clone Sequencing Purifying the DNA Template If you use E coli cells with a transformation efficiency of 1 x 10 cfu ug the LR reaction should give 75000 colonies if the entire LR reaction is transformed and plated 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 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 should not grow in the presence of chloramphenicol To confirm that your gene of interest is in frame with the N or C terminal peptide you may sequence your expression construct using the following priming sites if desired Refer to the diagrams on pages 9 10 for the locations of the primer binding sites for pEXP3 DEST and pEXP4 DEST Vector Forward Primer Reverse Primer pEXP3 DEST T7 Promoter primer 17 Reverse primer pEXP4 DEST T7 Promoter primer 17 Reverse primer For your convenience Invitrogen offers a custom primer synthesis service For more information go to www invitrogen com or contact Technical Support After you have generated the DNA template you must purify the DNA before proceeding to the prot
11. and down and incubate samples at 70 C for 10 minutes 4 Allow samples to cool for 1 2 minutes and centrifuge briefly at high speed in a microcentrifuge 5 Proceed to Adding Lumio In Gel Detection Enhancer below TM Add Lumio In Gel Detection Enhancer as described below 1 Thaw the Lumio In Gel Detection Enhancer and mix well by pipetting up and down TM Add 2 ul of Lumio Gel Detection Enhancer to each protein sample Mix samples well by pipetting up and down and incubate samples at room temperature for 5 minutes Return the Lumio In Gel Detection Enhancer to 20 C immediately after use 4 Load 5 20 ul of each sample on an appropriate gel and perform electrophoresis Be sure to run the gel long enough so that the dye front runs off the bottom of the gel to avoid masking smaller proteins 5 Proceed to Analyzing Lumio Tagged Proteins in Gels next page TM After the Lumio Green Detection Reagent and the Lumio Gel Detection Enhancer have been added to sample proceed to visualizing proteins within a few hours to avoid photobleaching 29 Analyzing Lumio Tagged Proteins in Gels Introduction Important Imager Specifications MORENO r B Va c o UV Exposure Time 26 After you have performed electrophoresis you can visualize Lumio tagged recombinant proteins directly in the gel General guidelines are provided below For more detailed information refer to the Lumio Gre
12. for expression S O C Medium LB agar plates containing the appropriate antibiotic to select for expression clones see previous page Add the following components to 1 5 ml microcentrifuge tubes at room temperature and mix Component Sample Negative Positive Control Control Entry clone 50 150 ng reaction 1 7 ul 1 7 ul pEXP3 DEST or pEXP4 DEST 1 pl 1 pl 1 pl 150 ng ul pENTR gus 50 ng ul 2 ul TE Buffer pH 8 0 to 8 ul to 8 ul 5 pl Remove the LR Clonase II enzyme mix from 20 C and thaw on ice for about 2 minutes Briefly vortex the LR Clonase II enzyme mix twice 2 seconds each time TM Add 2 ul of LR Clonase II enzyme mix to each sample and the positive control Do not add LR Clonase II enzyme mix to the negative control Mix well by pipetting up and down Reminder Return LR Clonase II enzyme mix to 20 C or 80 C immediately after use Incubate reactions at 25 C for 1 hour Note Extending the incubation time to 18 hours typically yields more colonies Add 1 ul of the Proteinase K solution to each reaction Incubate for 10 minutes at 37 C Transform 1 ul of the LR recombination reaction into a suitable E coli host follow the manufacturer s instructions and select for expression clones Note You may store the LR reaction at 20 C for up to 1 week before transformation if desired Continued on next page Performing the LR Recombination Reaction continued
13. pEXP4 DEST on page 10 for more information Continued on next page Generating an Expression Clone continued Recombination The recombination region of the expression clone resulting from pEXP3 DEST x Shaded regions correspond to those DNA sequences transferred from the entry clone into the pEXP3 DEST vector by recombination Non shaded regions Region of entry clone is shown below pEXP3 DEST Features of the Recombination Region are derived from the pEXP3 DEST vector Bases 194 and 1877 of the pEXP3 DEST sequence are marked T7 promoter priming site T7 promoter 1 GATCTCGATC CCGCGAAATT AATACGACTC ACTATAGGGA 81 151 1946 2026 HisG epitope GACCACAACG GTTTCCCTCT AGAAATAATT TTGTTTAACT Lumio tag Polyhistidine 6xHis region RBS Met His His His His His His Gly Ala Gly Gly Cys Cys Pro Gly Cys Cys im eee cl TTAAGAAGGA GATATACAT ATG CAT CAT CAC CAT CAC CAT GGT GCT GGT GGC TGT TGT CCT GGC TGT TGC TEV recognition site i 1 Gly Gly Gly Glu Asn Leu Tyr Phe Gln Gly Ile GGT GGC GGC GAA AAC CTG TAT TTT CAG GGA ATT TEV cleavage site 1877 NACCCAGCTT TCTTGTACAA AGTGGTTCGA TCGAAGCTTG NIGGGTCGAA AGAACATGTT TCACCAAGCT AGCTTCGAAC attB2 T7 reverse priming site 194 Ile Thr Ser Leu Tyz Lys Lys Ala Gly Pol ATC ACA AGT TTG TAC AAA AAA GCA GGC TNN TAG TGT TCA AAC ATG TTT TTT CGT CCG EE L atiB1 T7 transcription termination region I ATCCGGCTGC TAACAAAGCC CGAAAGGAAG CTG
14. www invitrogen com
15. 000 rpm Carefully remove the supernatant without disturbing the protein pellet Air dry for 2 3 minutes to allow excess acetone to evaporate Dilute the 4X Lumio Gel Sample Buffer included with the kit to 1X with deionized water Resuspend the pellet from Step 3 in 20 ul of 1X Lumio Gel Sample Buffer Proceed to Adding Lumio Detection Reagent next page Continued on next page Performing In Gel Lumio Detection continued Adding Lumio Detection Reagent Adding Lumio In Gel Detection Enhancer Important Add Lumio Detection Reagent to your protein sample as described below for in gel detection TM Important If you have already added Lumio Detection Reagent to your protein sample for real time detection page 19 skip this procedure and proceed TM directly to Adding Lumio In Gel Detection Enhancer below 1 Thaw the Lumio Green Detection Reagent and mix well by pipetting up and down 2 Add 02 pl of the Lumio Green Detection Reagent to each sample using a 2 ul pipetter e g a P2 pipetter Return the Lumio Green Detection Reagent to 20 C immediately after use Alternative If you do not have a P2 pipetter make a 1 5 dilution of the Lumio Green Detection Reagent using 1X Lumio Gel Sample Buffer Add 1 pl of this diluted Lumio Green Detection Reagent to each sample Diluted Lumio Green Detection Reagent is stable for up to 1 week at 20 C 3 Mix samples well by pipetting up
16. 23 20 Standard Protein Synthesis Introduction Incubation Conditions Materials to Have on Hand This section provides information on performing a standard Expressway Cell free E coli protein synthesis reaction without real time detection of Lumio tagged fusion proteins You can detect your Lumio tagged fusion proteins using in gel Lumio detection see page 23 following standard protein synthesis We recommend using an Eppendorf Thermomixer Fisher Catalog no 05 400 200 to shake your sample s at 37 C during the protein synthesis reaction If a thermomixer is unavailable you may use a standard shaking incubator or a standard shaking water bath We do not recommend using a non shaking incubator because it produces a less stable and less consistent temperature environment Provided by the user e Expression construct or other suitable DNA template purified resuspended in TE or water at a concentration greater than 500 ng ul S Methionine optional 3 000 Ci mmol 15 uCi ul Tubes or microplates suitable for use with your spectrofluorometer Thermomixer or standard shaking incubator see above RNase free pipette tips and microcentrifuge tubes Supplied with the kit Expressway E coli slyD Extract thaw on ice Expressway 2 5X IVPS Reaction Buffer A A thaw on ice Expressway 2X IVPS Feed Buffer thaw on ice T7 Enzyme Mix keep on ice store at 20 C after initial use 50 mM Amino Acids M
17. AGTTGGC TGCTGCCACC GCTGAGCAAT AACTAGCATA ACCCCTIGGG GAACTATATC CGGATCTGGC GCCTCTAAAC GGGTCTTGAG GGGTTTTTTG CTGAAAGGAG Continued on next page Generating an Expression Clone continued Recombination Region of pEXP4 DEST 61 1813 1861 1915 10 The recombination region of the expression clone resulting from pEXP4 DEST x entry clone is shown below Features of the Recombination Region Shaded regions correspond to those DNA sequences transferred from the entry clone into the pEXP4 DEST vector by recombination Non shaded regions are derived from the pEXP4 DEST vector Bases 105 and 1788 of the pEXP4 DEST sequence are marked A TGA stop codon is included downstream of the 6xHis tag to allow translation termination T7 promoter priming site d promoter GATCTCGATC CCGCGAAATT AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT 105 attB1 site I T AGAAATAATT TTGTTTAACT TTAAGAAGGA ATTATCAACA AGTTTGTACA AAAAAGCAGG TAATAGTTGT TCAAACATGT TTTTTCGTCC 105 attB2 sile m NE NAC CCA GCT TTC TTG TAC AAA GTG GTG ATC AAT TCG can SENE_ NTG GGT CGA AAG AAC ATG TTT CAC CAC TAG TTA AGC Pro Ala Phe Leu Tyr Lys Val Val Ile Asn Ser Lumio tag i 1 AAG CTT GAA GCT GGT GGC TGT TGT CCT GGC TGT TGC GGT GGC GGC ACC TTC GAA CTT CGA CCA CCG ACA ACA GGA CCG ACA ACG CCA CCG CCG TGG Lys Leu Glu Ala Gly Gly Cys Cys Pro Gly Cys Cys Gly Gly Gly Thr 6xHis tag l GGT CAT CAT CAC CAT CAC CAT
18. Green Detection Reagent is supplied pre complexed to the EDT 1 2 ethanedithiol which stabilizes and solubilizes the biarsenical reagent For more information on how the Lumio Green Detection Reagent binds to the Lumio tag see the Lumio Green Detection Kit manual The Lumio Green Detection Reagent binds a tetracysteine motif consisting of Cys Cys Xaa Xaa Cys Cys where Cys equals cysteine and Xaa equals any amino acid other than cysteine This motif is rarely seen in naturally occurring proteins allowing specific fluorescent labeling of recombinant proteins fused to the Lumio tag In the Lumio System the tetracysteine motif is Cys Cys Pro Gly Cys Cys as this motif has been shown to have a higher affinity for and more rapid binding to biarsenical compounds as well as enhanced stability compared to other characterized motifs Adams et al 2002 Continued on next page Lumio Technology continued Fluorescence Spectra Comparison of Real Time and In Gel Lumio Detection Methods The Lumio Green Detection Reagent has maximum excitation at 500 nm dye can also be excited in the UV region but with a lower efficiency and maximum emission at 535 nm see figure below This allows the detection of the Lumio fusion proteins using a UV transilluminator equipped with a standard camera or a visible light laser based scanner M 100 4 reus Excitation Emission co eo l O eo l
19. Invitrogen by technologies Expressway Lumio Cell Free Expression and Detection System For cell free protein synthesis and fluorescence detection of recombinant proteins containing the Lumio tag Catalog nos K9900 60 V960 03 V960 04 Rev Date 27 September 2011 Manual part no 25 0701 MANDO0000432 ii Table of Contents Kit Contents anid Storage sinan Aee a anre ree rne e i e eene e emen rete e e P rh EUER HER TR CER iv Additional Products nsn e tee e ote ere e eter ee NH eee eee eee eee ede ee eee vii Introduction ecc CPG 1 OVERVIEW 14e eh S e n To e PR PS ope pA 1 Lumio Technology ei esurire IAEA at iust Dates GS clo su adr EU POUR and D RR DU ae SE Pb a 4 Handling the Lumio Green Detection Keagenbaumecedisbeiase iut toile donee ke Ub ticae Eh bb does 6 Methods a2 CR 7 Creating an Expression Clone isis 2e fete I cess est inte dte hee erede sete sigesssteseneventuraseses 7 General Guidelines for Protein Synthesis continued sss 16 Protein Synthesis with Real Time Dumio Dele Chon ahaa tec eta te be o se a oie cal at re 17 otandard Protem Synthesis inasre ane da eU de ed Er er t ed etie e n aan 21 Performing In Gel Lumio Detectors a u ina ap derbi Med p ee E p b t ne EU b UR dee dh sonia duos 23 Analyzing Lumio Tagged Proteins in Gels iussi dede rdg e ua ut hd ete bl apad 26 Determining Protein Yield xe ee eee reet e n Ren e e e Sha Ire n t ee eee e dett 28 Purifying the Recombinant So
20. TGA GTTTGATCCG GCTGCTAACA AAGCCCGAAA CCA GTA GTA GTG GTA GTG GTA ACT Gly His His His His His His T7 reverse priming site T7 transcription termination region GGAAGCTGAG TTGGCTGCTG CCACCGCTGA GCAATAACTA GCATAACCCC TTGGGGCCTC Performing the LR Recombination Reaction Introduction E coli Host Antibiotic Selection LR Clonase II Enzyme Mix After you have obtained an entry clone containing your gene of interest you will perform an LR recombination reaction between the entry clone and either pEXP3 DEST or pEXP4 DEST and transform the reaction mixture into a suitable E coli host to select for an expression clone We recommend including the pENTR gus positive control supplied with the LR Clonase II enzyme mix in your experiments to help you evaluate your results You may use any recA end A E coli strain including TOP10 DH5o or equivalent for transformation Do not transform the LR reaction mixture into E coli strains that contain the F episome e g TOP10F These strains contain the ccd A gene and will prevent negative selection with the ccdB gene Both pEXP3 DEST and pEXP4 DEST contain the ampicillin resistance gene Expression clones may be selected using standard LB plates containing ampicillin at 100 ug ml The presence of the Zeocin resistance gene in pEXP4 DEST allows selection of E coli transformants using Zeocin antibiotic For selection use Low Salt LB agar plates containin
21. addition it allows detection of recombinant protein with the Anti HisG Antibodies Lumio tag Cys Cys Pro Gly Cys Cys Allows detection of the fusion protein by the binding of the biarsenical Lumio Green Detection Reagent Adams et al 2002 TEV recognition site Allows removal of the N terminal tag from your recombinant fusion protein using recombinant AcTEV Protease available from Invitrogen Catalog nos 12575 015 and 023 Carrington and Dougherty 1988 Dougherty et al 1988 attR1 and attR2 sites Bacteriophage derived DNA recombination sequences that permit recombinational cloning of the gene of interest from a Gateway entry clone Landy 1989 Chloramphenicol resistance gene Cm Allows counterselection of the plasmid ccdB gene Permits negative selection of the plasmid T7 transcription termination region Sequence from bacteriophage T7 that permits efficient transcription termination T7 reverse priming site Permits sequencing in the anti sense orientation f1origin Allows rescue of single stranded DNA bla promoter Allows expression of the ampicillin resistance gene Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pUC origin Permits high copy replication and maintenance in E coli Map and Features of pEXP4 DEST Map of pEXP4 The map below shows the elements of pEXP4 DEST DNA from t
22. al acetic acid for 15 30 minutes before drying If you have already destained the gel repeat destaining procedure Ethanol present in the protein synthesis reaction Make sure that any residual ethanol is removed during DNA purification Old pre cast gels Do not use pre cast gels after the expiration date Lumio Detection To troubleshoot Lumio Detection refer to the manual supplied with the kits or available from www invitrogen com 35 Appendix Recipes Low Salt LB 10 g Tryptone Medium with 5 g NaCl Zeocin 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 ml Adjust pH to 7 5 with 5 M NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cycle at 15 Ibs sq in and 121 C for 20 minutes Thaw Zeocin on ice and vortex before removing an aliquot Allow the medium to cool to at least 55 C before adding the Zeocin to 25 ug ml final concentration 5 Store plates at 4 C in the dark Plates containing Zeocin are stable for 1 2 weeks 36 Map and Features of pEXP3 DEST Map of pEXP3 The map below shows the elements of pEXP3 DEST DNA from the entry clone DEST replaces the region between bases 194 and 1877 The complete sequence for this vector available from www invitrogen com or by contacting Technical Support page 44 EP L3 Aro THEE STE cue PP eroded Comments for
23. ansform One Shot ccdB Survival 2 T1 Chemically Competent Cells Catalog 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 pEXP3 DEST is an N terminal fusion vector and contains an ATG initiation codon and a Shine Dalgarno ribosome binding site RBS with optimal spacing for proper translation in E coli Your gene of interest in the entry clone must Be in frame with the N terminal tag after recombination Contain a stop codon Refer to the diagram of the recombination region of pEXP3 DEST on page 9 for more information pEXP4 DEST is an C terminal fusion vector your gene in the entry clone must Contain an ATG initiation codon and a ribosome binding site RBS with optimal spacing for proper translation in E coli Note If you clone your gene of interest into an entry vector that supplies an RBS e g pENTR SD D TOPO then your gene of interest need only include an ATG initiation codon Not include a stop codon Be in frame with the C terminal tag after recombination Refer to the diagram of the recombination region of
24. ation see Using Unnatural Amino Acids below Add 1 volume of Feed Buffer containing Expressway 2X IVPS Feed Buffer and amino acids to the protein synthesis reaction after the initial 30 minute incubation Higher protein yields may be obtained by adding one half volume of Feed Buffer at 30 minutes and one half volume of Feed Buffer again at 2 hours after initiating the protein synthesis reaction Methionine is supplied separately in the kit to allow you to incorporate unnatural amino acids into your recombinant protein and adjust the amino acid concentration in the protein synthesis reaction Depending on your application you may use the following unnatural amino acids e Radiolabeled methionine Use S Methionine to produce radiolabeled protein for use in expression and purification studies See pages 19 and 21 for recommended amounts of labeled and unlabeled methionine e Heavy metal labeled methionine Use selenomethionine Budisa et al 1995 Doublie 1997 Hendrickson et al 1990 to produce labeled protein for use in X ray crystallographic studies See pages 19 and 21 for recommended amounts of labeled methionine Note When using selenomethionine do not use any unlabeled methioinine in the protein synthesis reaction e Do not store the EF coli slyD Extract 2 5X IVPS Reaction Buffer A A or 2X Feed Buffer at 20 C or room temperature as this may result in loss of activity e Freezing and thawing the E coli
25. ay 2 5X IVPS Reaction Buffer A A does not contain amino acids Store the entire box at 80 C or store individual components as listed below Item Amount Storage E coli slyD Extract 400 ul 80 C 2 5X IVPS Reaction Buffer A A 400 ul 80 C 2X IVPS Feed Buffer 500 pl 80 C T7 Enzyme Mix 20 ul 80 C 20 C after initial use DNase RNase Free Distilled Water 1 75 ml 20 C or 80 C Expressway Mini The following reagents are included in the Expressway Mini Amino Acids Amino Acids Module Store at 20 C Module Item Composition Amount Amino Acid Mix Methionine Contains all amino acids 50 mM 160 ul except for methionine in 50 mM HEPES pH 11 75 mM Methionine 75 mM in 50 mM HEPES 120 ul pH 7 5 4 mM DTT Vectors The Expressway Lumio vectors included with Cat nos K9900 70 K9900 90 V960 03 or V960 04 are listed below Store vectors at 20 C Item Composition Amount pEXP3 DEST 40 pL of 150 ng uL in 10 mM Tris 6 ug HCI 1 mM EDTA pH 8 0 pEXP3 GW CAT control 20 uL of 0 5 pg L in 10 mM Tris 10 ug HCI 1 mM EDTA pH 8 0 pEXP4 DEST 40 pL of 150 ng uL in 10 mM Tris 6 ug HCI 1 mM EDTA pH 8 0 pEXP4 ORF control 20 uL of 0 5 ug uL in 10 mM Tris 10 ug HCI 1 mM EDTA pH 8 0 Continued on next page Kit Contents and Storage continued Lumio Green Detection Kit Expressway Control Vector BenchMark
26. combinant protein using the Expressway Lumio Cell Free Expression and Detection System This section provides guidelines and a protocol to synthesize your protein RNase contamination may affect protein yield To reduce the chances of RNase contamination wear gloves and use RNase free reagents when performing the protein synthesis reaction To eliminate RNase from surfaces use RNase AWAY see page vii or a similar product The volume of the protein synthesis reaction may be scaled based on your needs For screening reactions the standard volume is 100 ul 50 ul initial reaction 50 ul Feed Buffer but this can be decreased to 25 ul reaction volume and increased up to 2 ml reaction volume Note that protein yields may vary depending on the nature of the protein expressed and the template used For a 100 ul protein synthesis reaction use 1 ug of template DNA plasmid or linear DNA For a 2 ml reaction use 10 15 ug of template DNA For optimal results purify DNA template before use see previous page Use a reaction vessel that contains a large enough surface area to allow moderate mixing to occur We recommend performing the 100 ul protein synthesis reaction in a sterile RNase free 1 5 ml tube If you are performing larger reaction volumes you may use sterile RNase free 50 ml conical tubes Other reaction vessels including 96 well 6 well or 12 well untreated culture plates are suitable e To obtain optimal prot
27. conducted by the buyer whether the buyer is an academic or for profit entity For licensing information for use in other than research please contact outlicensingGlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 45 Gateway Clone Distribution Policy Introduction Gateway Entry Clones Gateway Expression Clones Additional Terms and Conditions 46 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 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 als
28. ction Reagent supplied with the Lumio Green Detection Kit is a biarsenical compound and should be handled according to the guidelines provided in this section as well as in the Material Safety Data Sheet MSDS Exercise caution when handling the Lumio Green Reagent Wear protective clothing eyewear and gloves suitable for use with dimethyl sulfoxide e g nitrile gloves when handling the Lumio Green Detection Reagent Review the Material Safety Data Sheet MSDS before handling A dermal toxicity evaluation of the Lumio Green Detection Reagent was independently performed by MB Research Laboratories Spinnerstown PA USA by applying a full vial of material to the mouse skin In this study no adverse reaction or toxicity was noted Although arsenic compounds are toxic this product contains 0 276 of an organic arsenic compound and shows no toxicity at a maximum dose level likely to be handled The toxicology of this material however has not been fully investigated Handle according to your chemical hygiene plan and prevent contact with this material TM Treat accidental spills of the Lumio Green Detection Reagent on surfaces with 10 bleach for 10 minutes and then carefully clean up Discard arsenic containing waste according to your institution s guidelines Treat accidental contact of the Lumio Green Detection Reagent with human skin by washing excess reagent with soap and water as soon as possible Consult a physicia
29. duct Amount Catalog no Expressway N terminal Lumio Cell Free Expression 20 rxns K9900 70 and Detection System Expressway C terminal Lumio Cell Free Expression 20 rxns K9900 90 and Detection System Expressway Lumio Cell Free Expression and 20 rxns K9900 60 Detection System pEXP3 DEST Vector Kit 40 ul V960 03 pEXP4 DEST Vector Kit 40 ul V960 04 Amount based on 100 pl reaction size Kit Components following components The Expressway Lumio Cell Free Expression and Detection kits include the Component Catalog no K9900 70 K9900 90 K9900 60 V960 03 V960 04 Expressway Mini Expression Module Y y y Expressway Mini Amino Acid y Vy V Module Lumio Green Detection Kit N V y BenchMark Fluorescent Protein y Y V Standard Control Vector pEXP5 NT CALML3 Y pEXP3 DEST Vector Kit Y y pEXP4 DEST Vector Kit V y For a detailed description of the contents of the Expressway Modules see the next page For additional information on the Lumio Green Detection Kit refer to the manual supplied with the kit iv Kit Contents and Storage continued Shipping Storage The Expressway Lumio Cell Free Expression and Detection System components are shipped on dry ice Upon receipt store as described below and on the next page Expressway Mini The following reagents are included in the Expressway Mini Expression Expression Module Module Note that the Expressw
30. e 30 minute incubation prepare the Feed Buffer For each sample add the following reagents to a sterile RNase free microcentrifuge tube For multiple samples you may scale up the volume of reagents used accordingly and prepare one master mix Reagent Amount 2X IVPS Feed Buffer 25 ul 50 mM Amino Acids Met 1 25 pl 4 ul 75 mM Methionine 1ul 3pl DNase RNase free Distilled Water To final volume of 50 ul Note To generate radiolabeled protein using S methionine use 2 ul of 35S methionine and 1 ul unlabeled 75 mM methionine To generate labeled protein using selenomethionine use 2 ul of selenomethionine only do not add unlabeled methionine 4 After 30 minutes of incubation from Step 2 above add 50 ul of the Feed Buffer to the sample total volume 100 ul 5 Cap the tube and return the sample to the shaking incubator 300 rpm Incubate for up to 6 hours at 30 37 C as appropriate see page 14 To prepare proteins for in gel Lumio detection centrifuge and place the reaction on ice Proceed to Performing In Gel Lumio Detection next page 22 Performing In Gel Lumio Detection Introduction Recommended Gels BenchMark Fluorescent Protein Standard Lumio Gel Sample Buffer Lumio In Gel Detection Enhancer After you have synthesized your protein you are ready to prepare the protein samples for analysis with the Lumio Green Detection Kit To detect Lumio tagged fusion prote
31. egenerating system to provide an energy source for protein synthesis Kim et al 1996 Lesley et al 1991 Pratt 1984 Amino acids Met required for protein synthesis to occur and methionine provided separately for optimization of radiolabeling assays Optimized expression vectors with an N terminal pEXP3 DEST or C terminal pEXP4 DEST Lumio tag for specific detection of fusion proteins using the Lumio Green Detection Reagent The pEXP3 DEST and pEXP4 DEST vectors contain the necessary regulatory element in an optimal configuration for protein synthesis using the Expressway System See the detailed map and feature descriptions on pages 37 40 Bacteriophage T7 promoter for high level inducible expression of the recombinant protein of interest in the Expressway Systems or in E coli N or C terminal 6xHis tag for purification of recombinant fusion proteins N or C terminal Lumio tag for specific detection of recombinant proteins using Lumio Technology TEV recognition site for cleavage of the N terminal peptide from the recombinant fusion protein using TEV protease pEXP3 DEST only Two recombination sites att R1 and attR2 downstream of the T7 promoter for recombinational cloning of the gene of interest from an entry clone Chloramphenicol resistance gene Cm located between the two attR sites for counterscreening ccdB gene located between the atiR sites for negative selection Ampicillin resistance gene for selection
32. ein synthesis reaction You may use a variety of methods to purify your DNA template including commercial DNA purification kits For protocols to purify DNA refer to published reference sources Ausubel et al 1994 Sambrook et al 1989 When purifying your DNA template keep the following in mind For rapid isolation of high quality purified plasmid DNA we recommend using the PureLink HQ Mini Plasmid Purification Kit available from Invitrogen Other commercial DNA purification kits are suitable Do not gel purify your DNA template Purified DNA solution obtained from agarose gels significantly inhibits the protein synthesis reaction Ammonium acetate is not recommended for use in DNA precipitation as any residual contamination may inhibit translation Use sodium acetate Purified DNA must be free of RNases wear gloves and use RNase free reagents when preparing DNA Purified DNA should be free of excess ethanol or salt as both can inhibit translation Note Ethanol precipitated DNA should be carefully washed with 70 ethanol to remove excess salt and dried Purified DNA should be resuspended in 1X TE Buffer or water such that the final concentration is at a minimum of 500 ng ul 13 General Guidelines for Protein Synthesis Introduction Important Reaction Volumes Amount of DNA Template Reaction Vessel Incubation Conditions 14 After you have obtained purified template DNA you are ready to synthesize re
33. ein yield it is critical to mix the reaction thoroughly throughout the incubation period We recommend using a spectrofluorometer equipped with a thermomixer set to 1 200 rpm for real time protein synthesis or a shaking incubator set to 300 rpm Do not use stationary incubators such as incubator ovens or water baths as protein yields may be reduced by up to 30 5076 e Incubate the protein synthesis reaction at a temperature ranging from 30 C to 37 C The optimal temperature to use depends on the solubility of your recombinant protein and should be determined empirically Higher protein yields are generally obtained with incubation at higher temperatures i e 37 C however protein solubility generally improves with incubation at lower temperatures i e 30 C e You may obtain your protein of interest in as little as 1 5 hours of incubation after feeding 2 hours total Many reactions yield 80 90 of total protein within 2 hours However for maximum yield we recommend incubating the reaction for the full 6 hours Continued on next page General Guidelines for Protein Synthesis continued Amino Acid Concentration Feed Buffer Using Unnatural Amino Acids Handling Reagents Positive Control Use an amino acid concentration ranging from 1 mM to 4 mM in the protein synthesis reaction The recommended amino acid concentration is 1 25 mM each but may be adjusted according to the protein being synthesized and your applic
34. emplate DNA in a 2 ml protein synthesis reaction e If you are expressing a large protein increase the amount of DNA template used in the protein synthesis reaction to 20 pg 32 Continued on next page Troubleshooting continued Synthesizing Proteins continued Problem Reason Solution Low or no yield of protein but control reaction produces protein continued Sample incubated in a non shaking incubator or spectrofluorometer without a mixer e If you are not performing real time Lumio detection use a thermomixer 1 400 rpm or a shaking incubator 275 325 rpm or a water bath see page 21 e For real time Lumio detection use a spectrofluorometer with an incubator and mixer or use a recommended alternative page 19 Insufficient feeding e Add one volume of Feed Buffer to the sample i e 1 ml Feed Buffer to 1 ml sample 30 minutes after initiating protein synthesis Add one half volume of Feed Buffer to the sample i e 25 ul Feed Buffer to 50 ul sample 30 minutes and 2 hours after initiating protein synthesis Large protein being expressed Protein yield may decrease as the size of the protein increases optimize expression conditions e Reduce incubation temperature to 25 C 30 C during protein synthesis Expression time too short Extend expression time up to 4 hours Protein forms aggregates Reduce the incubation temperature to 25 C 30
35. en Detection Kit manual Detection of recombinant proteins with the Lumio Green Detection Kit is not permanent and is lost by subsequent staining of the gel with other protein stains and western blotting It is extremely important to record a permanent image of the gel prior to staining the gel with protein stains and gel drying For optimal visualization of the fluorescent protein bands after detection with Lumio Green Detection Kit you will need e Animager equipped with a UV transilluminator 302 or 365 nm a standard camera or CCD camera and an ethidium bromide filter SYBR Green filter or band pass filter encompassing the emission maxima 535 nm of the stain Note If you are using a 365 nm UV transilluminator you may have to expose the gel for a longer time as the sensitivity is lower than a 302 nm UV transilluminator OR e A laser based scanner with a laser line that falls within the excitation maxima of the stain 500 nm a 535 nm long pass filter or a band pass filter center near the emission maxima of 535 nm The sensitivity of detection is more with laser based scanners equipped with appropriate filters than with UV transillumination e If you are using pre cast gels in cassettes and are performing imaging with a UV transilluminator we recommend removing the gel from the cassette after electrophoresis is complete Avoid touching the gel with bare hands while handling or imaging the gel e If you are imagin
36. essway Lumio Cell Free Expression and Detection System is covered under the licenses detailed below The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 This product is licensed from Hoffmann LaRoche Inc Nutley NJ and Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from OIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany ccdB selection technology is described in Bernard et al Positive Selection Vectors Using the F Plasmid ccdB Killer Gene Gene 148 1994 71 74 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
37. et Note When thawing the 50 mM Amino Acids Met the solution may have a brown or yellowish tint This is normal and does not affect the activity of the amino acids 75 mM Methionine DNase RNase free distilled water e Control plasmid optional resuspended to 0 5 ug pl in sterile water Continued on next page 21 Standard Protein Synthesis continued Performing Use the protocol below to synthesize your protein from the DNA template Standard Protein without real time Lumio detection Synthesis 1 For each sample add the following reagents to the appropriate reaction vessel on ice For multiple samples scale up the volume of each reagent accordingly and aliquot the cocktail into individual tubes wells Reagent Amount E coli slyD Extract 20 ul 2 5X IVPS Reaction Buffer A A 20 ul 50 mM Amino Acids Met 1 25 ul 4 ul 75 mM Methionine 1ul 3gl 17 Enzyme Mix 1ul DNA Template 1ug DNase RNase free Distilled Water To a final volume of 50 ul Note To generate radiolabeled protein using S methionine use 2 ul of 35S methionine and 1ul unlabeled 75 mM methionine To generate labeled protein using selenomethionine use 2 ul of selenomethionine only do not add unlabeled methionine 2 Close the tube and incubate sample in a standard shaking incubator 300 rpm at 30 C for 30 minutes If the protein you are synthesizing is known to be soluble you may incubate the sample at 37 C 3 During th
38. g 25 ug ml Zeocin see page 36 for a recipe Note that for Zeocin to be active the salt concentration of the bacterial medium must remain low 90 mM and the pH must be 7 5 Zeocin is available from Invitrogen see page vii for ordering information Instructions to prepare and handle Zeocin are supplied with the product Gateway LR Clonase II enzyme mix Catalog no 11791 020 combines the proprietary enzyme formulation and 5X LR Reaction Buffer previously supplied as separate components in Gateway LR Clonase enzyme mix into an optimized single tube format to allow easier set up of the LR recombination reaction Use the protocol provided on the next page to perform the LR recombination reaction using LR Clonase II enzyme mix Continued on next page 11 Performing the LR Recombination Reaction continued Materials Needed Performing the LR Reaction 12 Purified plasmid DNA of your entry clone 50 150 ng UII in F pH 8 0 pEXP3 DEST or pEXP4 DEST 150 ng l in TE pH 8 0 LR Clonase II enzyme mix Invitrogen Catalog no 11791 020 keep at 20 C or 80 C until immediately before use pENTR gus positive control optional 50 ng ul in TE pH 8 0 supplied with the LR Clonase II enzyme mix TE Buffer pH 8 0 10 mM Tris HCl pH 8 0 1 mM EDTA IM 2 1g Proteinase K solution supplied with the LR Clonas ll enzyme mix thaw and keep on ice until use Appropriate competent E coli host and growth media
39. g with a laser based scanner you do not have to remove the gel from the cassette If you are using an imager with UV transilluminator be sure to adjust the settings and filters on the imager prior to turning on the UV light The fluorescent dye of the Lumio Green Reagent is sensitive to photobleaching We recommend a 4 10 second exposure Avoid exposing the gel to UV light for a long time You should see fluorescent bands of Lumio fusion proteins and the gel should have minimal background Note The Lumio fusion protein bands appear white or black depending on the type of imaging system used for imaging the gels Continued on next page Analyzing Lumio Tagged Proteins in Gels continued Assay for CAT Note What to Do Next If you use the pEXP3 GW CAT positive control vector you can assay for CAT protein using CAT Antiserum available from Invitrogen see page vii for ordering information Other commercial kits are available for assaying CAT expression The molecular weight of the CAT fusion protein is approximately 30 kDa The N and C terminal peptide containing the Lumio tag and 6xHis tag will add approximately 3 5 kDa to your protein Once you have verified expression you may use the recombinant protein in any downstream application of your choice If you plan to use the recombinant protein for structural analyses including x ray crystallography note that you must purify the recombinant protein bef
40. gned for in vitro transcription and translation of target DNA to protein in a single tube with the ability to perform both real time and rapid in gel detection of Lumio tagged recombinant proteins Using this kit your gene of interest is fused to the Lumio tag enabling sensitive and specific in gel detection of the Lumio tagged fusion protein in polyacrylamide gels without the need for staining or western blotting You can also monitor real time synthesis of the Lumio tagged fusion protein using a standard fluorometer The system uses an E coli extract and a T7 Enzyme Mix that have been optimized for expressing full length active protein from DNA constructs in about 2 hours Proteins with the Lumio tag can be detected during synthesis using real time detection and after synthesis using in gel detection The Expressway Cell Free E coli Expression System uses an optimized E coli extract a reaction buffer containing an ATP regenerating system and amino acids to allow high level synthesis of your recombinant protein of interest At one or several time points after initiating the protein synthesis reaction the reaction is supplemented with an optimized Feed Buffer containing a proprietary mixture of salts amino acids and other substrates that are depleted or degraded over time during protein synthesis see Figure below Addition of this Feed Buffer to the reaction replenishes these components and allows continuous cell free protein syn
41. he certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price 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 The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Life Technologies makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have 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 Research Use Only Limited Use Label License Limited Use Label License ULB ccadB Selection Technology Use of the Expr
42. he entry clone DEST replaces the region between bases 105 and 1788 The complete sequence for this vector is available from www invitrogen com or by contacting Technical Support page 44 Ep atfR Cm ccdB attR2 Lumio tag exHis Comments for pEXP4 DEST 4415 nucleotides T7 promoter bases 20 36 T7 promoter priming site bases 20 39 attR1 site bases 98 222 Chloramphenicol resistance gene CmP bases 331 990 ccdB gene bases 1332 1637 attR2 site bases 1678 1802 Lumio tag bases 1831 1848 Polyhistidine 6xHis region bases 1864 1881 T7 reverse priming site bases 1935 1954 T7 transcription termination region bases 1940 2024 Zeocin resistance gene bases 2168 2542 Ampicillin resistance gene bases 2563 3423 pUC origin bases 3568 4241 Continued on next page 39 Map and Features of pEXP4 DEST continued Features of pEXP4 DEST 40 The pEXP4 DEST vector 4415 bp contains the following elements Features have been functionally tested and the vector fully sequenced Feature Benefit T7 promoter Allows high level inducible expression of your recombinant protein in the Expressway Systems or in E coli strains expressing the T7 RNA polymerase T7 promoter priming site Allows sequencing in the sense orientation attR1 and attR2 sites Bacteriophage derived DNA recombination sequences that permit recombinational cloning of the gene of interest from a Gateway entry clo
43. he tubes microplate to a spectrofluorometer and collect excitation and emission data Let the program run for 30 minutes and during this time prepare the Feed Buffer For each sample add the following reagents to a sterile RNase free microcentrifuge tube For multiple samples you may scale up the volume of reagents used accordingly and prepare one master mix Reagent Amount 2X IVPS Feed Buffer 25 ul 50 mM Amino Acids Met 1 25 ul 4 ul 75 mM Methionine 1gul 3u Lumio Green Detection Reagent 1 ul DNase RNase free Distilled Water To final volume of 50 ul Note To generate radiolabeled protein using S methionine use 2 ul of 9S methionine and 1 ul unlabeled 75 mM methionine To generate labeled protein using selenomethionine use 2 ul of selenomethionine only do not add unlabeled methionine After 30 minutes of incubation from Step 5 above add 50 ul of the Feed Buffer to the samples total volume 100 ul between data time points Note You may incubate the reaction for up to 6 hours to obtain greater protein yield You may also incubate at temperatures as low as 25 C to decrease the rate of protein synthesis and to promote proper folding If you will be incubating tubes at temperatures lower than 37 C we recommend extending the incubation time to 4 hours To prepare proteins for in gel Lumio detection briefly centrifuge and place the reaction on ice Proceed to Performing In Gel Lumio Detection page
44. ii Additional Products continued Products to Purify If you have expressed your protein of interest in frame with the N or C terminal Recombinant Fusion Protein below for ordering information polyhistidine 6xHis tag you may use a nickel charged agarose resin such as ProBond or Ni NTA to purify your recombinant fusion protein See the table Product Quantity Catalog no ProBond Purification System 6 purifications K850 01 ProBond Nickel chelating Resin 50 ml R801 01 150 ml R801 15 Ni NTA Purification System 6 purifications K950 01 Ni NTA Agarose 10 ml R901 01 25 ml R901 15 Purification Columns 50 R640 50 10 ml polypropylene columns AcTEV Protease pEXP3 DEST only 1 000 units 12575 015 Expressway Kits Other Expressway Cell Free E coli Expression System Modules are available separately from Invitrogen Ordering information is provided below For more information go to www invitrogen com or contact Technical Support page 44 Product Quantity Catalog no Expressway Mini Cell free E coli Expression System 1 kit K9901 00 Expressway Maxi Cell free E coli Expression System 1 kit K9900 97 Expressway Maxi Cell free E coli Expression System 1 kit K9900 96 with pEXP5 TOPO vectors viii Overview Introduction How the System Works Applications Introduction The Expressway Lumio Cell Free Expression and Detection System is desi
45. in E coli pUC origin for high copy replication and maintenance of the plasmid in E coli All Invitrogen Expressway System Kits contain an optimized E coli slyD extract The slyD extract promotes the high yield expression of full length active protein from DNA constructs under the reaction conditions specified in this manual Overview continued Other Expressway Systems Lumio Technology Gateway Technology Experimental Outline If you have used other Invitrogen Expressway Cell Free E coli Expression Systems note that some of the components including the Expressway IVPS E coli Extract and the Expressway 2 5X IVPS E coli Reaction Buffer supplied with older Expressway kits contain different formulations and may not be compatible with this system For optimal results use the components supplied in this kit to perform the protein synthesis reaction The Lumio System is based on the FIAsH Fluorescein Arsenical Hairpin technology which uses a biarsenical reagent to bind and detect proteins containing a tetracysteine motif i e Lumio tag Griffin et al 1998 The biarsenical reagent becomes strongly fluorescent only upon binding to the Lumio tag allowing specific detection of Lumio tagged recombinant proteins from endogenous proteins in gels or during real time protein synthesis For more information about Lumio Technology see page 4 and the Lumio Green Detection Kit manual Gateway is a
46. ins by polyacrylamide gel electrophoresis you must first precipitate the proteins with acetone to remove background smearing and add the Lumio Gel Sample Buffer and Lumio In Gel Detection Enhancer If you have not already added Lumio Green Detection Reagent for real time detection you will also add this reagent General guidelines and protocols are provided below For more detailed information refer to the Lumio Green Detection Kit manual If you have performed trace labeling using S Methionine you may use TCA precipitation to determine the amount of radiolabeled methionine incorporated and to calculate the yield of protein see Determining Protein Yield page 28 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 For more information about pre cast gels available go to www invitrogen com or contact Technical Support see page 44 The BenchMark Fluorescent Protein Standard also available separately as cat no LC5928 allows easy and direct visualization of molecular weight ranges of your Lumio fusion protein on a SDS PAGE gel For detailed information and specifications refer to the BenchMark Fluorescent Protein Standard manual The BenchMark Fluorescent Protein Standard proteins are easily detected using a UV tra
47. ker Always use the Lumio Gel Sample Buffer 4X to prepare samples for electrophoresis Wear protective clothing eyewear and gloves suitable for use with dimethyl sulfoxide e g nitrile gloves when handling the Lumio Green Detection Reagent Use the Lumio Gel Sample Buffer 4X in a certified fume hood Use an appropriate percentage of acrylamide gel that will best resolve your protein of interest Visualize the gel immediately after electrophoresis to prevent diffusion of proteins as the proteins are not fixed in the gel during Lumio detection Avoid touching the gel with bare hands while handling or imaging the gel Proteins that DO NOT contain Lumio Green Detection Reagent may be stored in 1X Lumio Gel Sample Buffer at 20 C Protein synthesis reaction from page 19 or page 22 Acetone at 20 C 4X Lumio Gel Sample Buffer supplied with the kit Lumio Green Detection Reagent if not already added for real time detection supplied with the kit Lumio In Gel Detection Enhancer supplied with the kit Water bath or heat block set at 70 C Freezer set at 20 C Appropriate pre cast gels and running buffer Use the following protocol to prepare your proteins for gel electrophoresis 1 To 5 ul of the protein synthesis reaction product from page 19 or page 22 add 20 ul of cold acetone 20 C Mix well Incubate at 20 C for 20 minutes Centrifuge for 5 minutes at room temperature in a microcentrifuge at 12
48. luble Fusion Protein ssssseeeeeeeeeeeee nennen 30 Sample Protein Synthesis Experiment entente ren nre eb e e ndis 31 Troubleshooting 1r ee ERU euh io etre Cb Age Ho tero iro then tete et be o aspe Ere oodd 32 PN ooa D Qum ELS RULES 36 RECIPES p e A en 36 Map and Features of pEXP9 DBOT 4rd edet mete nnper re ttes iei pad ee e teret n 37 Map and Features of pEXPA DEST tette tte ti e erre ette n re eie dere reri e PR EG 39 Map ot pEXP9 GW CAT tees aesti t erede ete A e re ote a c Re tie ee ee 41 Map OE pEXUA ORE s t tet tete ede d e ttm LT b i t e d cts 42 Map and Features of pEXP5 NT CALMLG sees eene een nenne nennen enne nnnnnnnnnenenennn 43 Technical Supports n eee e eet tte b i ir tete CURA ederet tke E Go her eie cell oed EE BEL DL 44 Purchaser Notficatiob cai Aere S EE Ne bite i Heise t bee it o eet brote Eben 45 Gateway Clone Distribution Policy au ioter eet pi leote Ato e eR ea neon oti Seed EE oem oae 46 References x aste aedi eee te deii rete t e tere E aree ere Hee eate ETE quud 47 iii Kit Contents and Storage Kits have been reconfigured New reagents are supplied and quantities of Important reagents are different If you have used these products in the past discard old versions of this manual and use the instructions provided in this manual Types of Kits This manual is supplied with the following products Pro
49. molecular weight of protein 10 29 Purifying the Recombinant Soluble Fusion Protein Introduction Note ProBond and Ni NTA Guidelines for Purification 30 The presence of the N terminal or C terminal 6xHis tag in the pEXP3 DEST and pEXP4 DEST vectors allows purification of your recombinant protein with a metal chelating resin such as ProBond or Ni NTA see page viii for ordering information This section provides guidelines for purification The pEXP3 DEST vector contains a Tobacco Etch Virus TEV recognition site to allow removal of the N terminal tag from your recombinant fusion protein using TEV protease See page vii for additional products ProBond and Ni NTA are nickel charged agarose resins that can be used for affinity purification of fusion proteins containing the 6xHis tag Proteins bound to the resin may be eluted with either low pH buffer or competition with imidazole or histidine e To purify your fusion protein using ProBond or Ni NTA follow the guidelines below and detailed instructions included with each product You may download the appropriate manuals from www invitrogen com e To purify your fusion protein using another metal chelating resin refer to the manufacturer s instructions Follow these guidelines when purifying your recombinant fusion protein using ProBond or Ni NTA Remember to use criteria appropriate for purification under native conditions For details
50. n following contact with Lumio Green Reagent Do not treat arsenic skin exposure with EDT 1 2 ethanedithiol as this may promote uptake of the Lumio Green Reagent into the body All excess reagents that contain or have come in contact with arsenic compounds should be discarded according to your institution s guidelines and all applicable local state and federal requirements In general we recommend disposing of protein samples labeled with the Lumio Green Detection Reagent and polyacrylamide gels containing protein samples labeled with the Lumio Green Detection Reagent as hazardous waste For specific disposal requirements in your area consult your safety officer Methods Creating an Expression Clone Introduction DNA Templates Generating an Entry Clone Destination Vectors The pEXP3 DEST vector 4 6 kb and pEXP4 DEST vector 4 4 kb are designed to allow T7 based high level expression of N and C terminal tagged recombinant fusion proteins using the Expressway Lumio Cell Free Expression and Detection System Vectors are derived from Invitrogen s pEXP1 DEST vector and adapted for use with Lumio Technology For a vector map and features see pages 37 40 pEXP3 DEST allows you to fuse Lumio and 6xHis tags to the N terminus of your protein of interest using Gateway Technology for production of recombinant fusion proteins that can be easily detected and purified The following DNA templates may be u
51. ne Landy 1989 Chloramphenicol resistance gene Cm Allows counterselection of the plasmid ccdB gene Allows negative selection of the plasmid TM Lumio tag Cys Cys Pro Gly Cys Cys Allows detection of the fusion protein by the binding of the biarsenical Lumio Green Detection Reagent Adams et al 2002 Polyhistidine 6xHis tag Allows purification of recombinant fusion protein on metal chelating resin e g ProBond or Ni NTA In addition allows detection of recombinant protein with the Anti His C term Antibodies T7 reverse priming site Allows sequencing in the anti sense orientation T7 transcription termination region Sequence from bacteriophage T7 that permits efficient transcription termination Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pUC origin Allows high copy replication and maintenance in E coli Map of pEXP3 GW CAT Description pEXP3 GW CAT is a 3651 bp control vector expressing the chloramphenicol acetyltransferase CAT protein The CAT gene is cloned in optimal configuration for expression using the Expressway Lumio Cell Free Expression and Detection System The molecular weight of the CAT fusion protein is approximately 30 kDa Map of pEXP3 The map below shows the elements of pEXP3 GW CAT The complete sequence GW CAT of the vector is available from www invitrogen com
52. ng real time analysis 17 Protein Synthesis with Real Time Lumio Detection continued Materials Needed 18 Provided by the user e Expression construct or other suitable DNA template purified resuspended in TE or water at a concentration greater than 500 ng ul S Methionine optional 3 000 Ci mmol 15 Ci ul Tubes or microplates suitable for use with your spectrofluorometer Spectrofluorometer equipped with an incubator and mixer see next page RNase free pipette tips and microcentrifuge tubes Supplied with the kit Expressway E coli slyD Extract thaw on ice Expressway 2 5X IVPS Reaction Buffer A A thaw on ice Expressway 2X IVPS Feed Buffer thaw on ice T7 Enzyme Mix keep on ice store at 20 C after initial use 50 mM Amino Acids Met Note When thawing the 50 mM Amino Acids Met the solution may have a brown or yellowish tint This is normal and does not affect the activity of the amino acids 75 mM Methionine DNase RNase free distilled water e pEXP3 GW CAT or pEXP4 ORF control plasmid optional resuspended to 0 5 ug ul in sterile water Note The pEXP5 NT CALML3 control plasmid supplied with cat no K9900 60 is not a Lumio fusion vector and should not be used as a control for real time Lumio detection e Lumio Green Detection Reagent Note The color of the Lumio Green Detection Reagent may change from clear to pink during storage This will not affect the performance of
53. nsilluminator or a visible light laser based scanner at the same excitation and emission wavelengths as your Lumio fusion protein The standard consists of seven distinct protein bands in the range of 11 155 kDa and is supplied in a ready to use format The recommended loading volume is 5 ul The Lumio Gel Sample Buffer 4X supplied with the kit is a proprietary sample buffer containing protein denaturing and reducing agents The buffer is specifically formulated to provide optimal results with the Lumio Green Detection Reagent Always use the Lumio Gel Sample Buffer 4X to prepare samples for electrophoresis To prevent oxidation of the reducing agent in the buffer store the Lumio Gel Sample Buffer 4X at 20 C and minimize exposure to air Use the buffer immediately upon removal from 20 C and return the buffer to 20 C immediately after use TM The Lumio In Gel Detection Enhancer is a proprietary solution and is designed to reduce the non specific binding of Lumio Green Detection Reagent with endogenous proteins Continued on next page 23 Performing In Gel Lumio Detection continued l NOMEN v B Va o V Materials Needed Preparing Expressed Proteins 24 For optimal results with the Lumio Green Detection Kit follow these guidelines Load at least 1 picomole of the Lumio fusion protein Use 5 ul of BenchMark Fluorescent Protein Standard on a mini gel as a molecular weight mar
54. o 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 References Adams S R Campbell R E Gross L A Martin B R Walkup G K Yao Y Llopis J and Tsien R Y 2002 New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo Synthesis and Biological Applications J Am Chem Soc 124 6063 6076 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 Budisa N Steipe B Demange P Eckerskorn C Kellermann J and Huber R 1995 High Level Biosyn
55. ontrol reaction produces protein Low or no yield of protein DNA template not optimally configured e Make sure that the ATG initiation codon is in the proper context for expression i e check spacing and placement after the RBS e Fusion of your protein to an N or C terminal tag may affect RNA structure and lower translation levels Try moving the fusion tag to the other terminus using either pEXP3 DEST or pEXP4 DEST Gene of interest not cloned in frame with the N or C terminal tag Generate a new expression construct making sure that your gene of interest is cloned in frame with the N or C terminal tag confirm by sequencing Incorrect LR recombination reaction protocol used The LR reaction protocol provided on page 12 is for use with LR Clonase II enzyme mix only If you are using LR Clonase enzyme mix refer to the protocol provided with the product DNA template not pure e Contaminated with ethanol sodium salt or ammonium acetate e Contaminated with RNases e Prepare new DNA template taking care to remove excess ethanol and or salt after precipitation e Do not use ammonium acetate to precipitate DNA Use sodium acetate e Wear gloves and use RNase free reagents when preparing DNA DNA template purified from agarose gel Do not purify your DNA from a gel See the purification guidelines on page 13 Insufficient amount of DNA template used e Use 10 15 ug of t
56. or by contacting Technical Support page 44 EP EJ Aro See CAT TM IIT Comments for pEXP3 GW CAT 3651 nucleotides T7 promoter priming site bases 20 39 Ribosome binding site bases 85 92 Initiation ATG bases 100 102 Polyhistidine 6xHis region bases 103 120 Lumio tag bases 133 150 TEV recognition site bases 160 180 attB1 site bases 187 211 CAT gene bases 232 891 attB2 site bases 911 935 T7 transcription termination region bases 956 1085 T7 reverse priming site bases 995 1014 f1 origin bases 1156 1611 bla promoter bases 1698 1796 Ampicillin bla resistance gene bases 1797 2657 pUC origin bases 2802 3475 41 Map of pEXP4 ORF Description Map of pEXP4 ORF 42 pEXP4 ORF is a 3920 bp control vector expressing a human open reading frame and was generated using the LR recombination reaction between an Invitrogen Ultimate hORF entry clone and pEXP4 DEST The molecular weight of the native protein is approximately 43 kDa The molecular weight of the protein fused to the C terminal tag is approximately 46 5 kDa The map below shows the elements of pEXP4 ORF The complete sequence of the vector is available from www invitrogen com or by contacting Technical Support page 44 E EXE Lumio tag DAE Comments for pEXP4 ORF 3920 nucleotides T7 promoter bases 20 36 T7 promoter priming site bases 20 39 attB1 site bases 98 122 Control ORF bases 139 1284 attB2 site ba
57. ore use Use any method of choice to purify your recombinant protein If you have expressed your recombinant protein with the N terminal or C terminal 6xHis tag you may purify your recombinant protein using a metal chelating resin such as ProBond or Ni NTA For guidelines to purify recombinant protein using ProBond or Ni NTA see page viii Note Other metal chelating resins are suitable 27 Determining Protein Yield Introduction Determining Total Counts Performing TCA Precipitation 28 If you have included radiolabeled methionine in the protein synthesis reaction you may use TCA precipitation to determine the amount of radiolabeled methionine incorporated and to calculate the yield of protein Mix and spot 5 ul of each radiolabeled reaction from page 19 or page 22 ona glass microfiber filter Type GF C Whatman Catalog no 1822 021 Set aside and let dry Do not wash or TCA precipitate these filters Place the filters in scintillation vials and add scintillation fluid Count samples in a scintillation counter A protocol is provided below to perform TCA precipitation using a vacuum filtration device e 2 Millipore 1225 Sampling Manifold or similar Performing TCA Precipitation Using a Vacuum Filtration Device 1 Aliquot 5 ul of each radiolabeled reaction from page 19 or page 22 into separate glass tubes Add 100 ul of 1 N NaOH to each reaction and incubate for 5 minutes at room temperature
58. orming protein synthesis with real time detection of Lumio tagged proteins Note that real time signal strength does not correlate to protein expression levels so performing in gel detection is recommended in addition to real time detection Real time detection of Lumio tagged proteins allows you to directly monitor production of your recombinant proteins during protein synthesis using a standard spectrofluorometer To perform real time detection simply add Lumio Green Detection Reagent to your protein synthesis reaction and incubate the reaction in a spectrofluorometer programmed to measure fluorescence at specified time points Real time detection of Lumio tagged proteins is NOT a direct quantitative method to determine protein concentration Depending on the folding conformation of the protein the Lumio tag may be buried within folded regions thereby preventing binding of the detection reagent or the expressed protein itself may quench the fluorescence The fluorescent signal generated does not necessarily correspond to the amount of protein synthesized and should be confirmed using in gel detection page 24 following real time analysis You can perform in gel Lumio detection following real time detection with no loss of fluorescent intensity and without the use of additional Lumio Green Detection Reagent Refer to the section on in gel Lumio detection on page 24 for instructions on preparing for in gel detection followi
59. pEXP3 DEST 4607 nucleotides T7 promoter priming site bases 20 39 Ribosome binding site bases 85 92 Initiation ATG bases 100 102 Polyhistidine 6xHis region bases 103 120 Lumio tag bases 133 150 TEV recognition site bases 160 180 attR1 site bases 187 311 Chloramphenicol resistance gene Cm bases 420 1079 ccdB gene bases 1421 1726 attR2 site bases 1767 1891 T7 transcription termination region bases 1912 2041 T7 reverse priming site bases 1951 1970 f1 origin bases 2112 2567 bla promoter bases 2654 2752 Ampicillin b a resistance gene bases 2753 3613 pUC origin bases 3758 4431 Continued on next page 37 Map and Features of pEXP3 DEST continued Features of pEXP3 DEST 38 been functionally tested The pEXP3 DEST vector 4607 bp contains the following elements Features have Feature Benefit 17 promoter Permits high level inducible expression of your recombinant protein in the Expressway Systems or in E coli strains expressing the T7 RNA polymerase T7 promoter priming site Allows sequencing in the sense orientation Ribosome binding site Optimally spaced from the initiation ATG for efficient translation of PCR product Initiation ATG Allows translation initiation of the recombinant fusion protein N terminal polyhistidine 6xHis tag Permits purification of recombinant fusion protein on metal chelating resin e g ProBond or Ni NTA In
60. quot the cocktail into individual tubes wells Reagent Amount E coli slyD Extract 20 ul 2 5X IVPS Reaction Buffer A A 20 ul 50 mM Amino Acids Met 1 25 ul 4 ul 75 mM Methionine 1ul 3pl 17 Enzyme Mix 1ul DNA Template 1ug Lumio Green Detection Reagent 1 ul DNase RNase free Distilled Water To a final volume of 50 ul Note To generate radiolabeled protein using S methionine use 2 ul of 35S methionine and 1 ul unlabeled 75 mM methionine To generate labeled protein using selenomethionine use 2 ul of selenomethionine only do not add unlabeled methionine 3 Program your spectrofluorometer for a 2 hour up to 6 hour incubation at 37 C with mixing Program fluorescence data collection at specified time points e g every 10 minutes See above for wavelength settings for Lumio detection Protocol continues on next page Continued on next page 19 Protein Synthesis with Real Time Lumio Detection continued Performing Continued from the previous page Protein Synthesis 4 with Real Time Lumio Detection continued After programming the spectrofluorometer insert your sealed tubes or microplate and run the program Alternative If your spectrofluorometer does not include an incubator or mixer you can incubate the tubes or microplate at 37 C for 2 hours in a thermomixer set at 14 000 rpm or shaking incubator set at 275 325 rpm At regular intervals e 2 10 minutes transfer t
61. refer to the ProBond or Ni NTA manual as appropriate 1 Prepare the purification column containing ProBond or Ni NTA agarose resin After applying the resin to the purification column wash with 4 volumes of water followed by 8 volumes of Binding Buffer supplied with the kit 50 mM NaPO pH 8 0 500 mM NaCl to equilibrate the column 2 Optional applies only to protein synthesis reactions containing extra components e g detergents chaperones other than those supplied with the Expressway kit Dilute the Expressway reaction from page 19 or page 22 1 1 with Binding Buffer 50 mM NaPO pH 8 0 500 mM NaCl 3 Centrifuge the reaction at 15 000 x g for 10 minutes at room temperature to remove insoluble material 4 Load the supernatant containing soluble protein onto the equilibrated resin and incubate i e batch binding for 30 minutes at the desired temperature 5 Wash the column twice with 2 volumes of Binding Buffer each time 6 Wash the column twice with 2 volumes of Binding Buffer containing 20 mM imidazole 7 Elute the protein using an Elution buffer containing an appropriate amount of imidazole e g 250 mM imidazole 8 Analyze the fractions using SDS PAGE 9 Poolthe desired fractions and dialyze if necessary Sample Protein Synthesis Experiment Introduction This section provides an example of a typical protein synthesis experiment performed using the Expressway Lumio Cell Free Expression and
62. s of Lambda Site specific Recombination Ann Rev Biochem 58 913 949 Lesley S A Brow M A and Burgess R R 1991 Use of in vitro Protein Synthesis from Polymerase Chain Reaction generated Templates to Study Interaction of Escherichia coli Transcription Factors with Core RNA Polymerase and for Epitope Mapping of Monoclonal Antibodies J Biol Chem 266 2632 2638 Pratt J M 1984 Transcription and Translation Oxford S J IRL Press continued on next page 47 References continued Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Studier F W Rosenberg A H Dunn J J and Dubendorff J W 1990 Use of 17 RNA Polymerase to Direct Expression of Cloned Genes Meth Enzymol 185 60 89 Zubay G 1973 In vitro Synthesis of Protein in Microbial Systems Annu Rev Genet 7 267 287 2011 Life Technologies Corporation All rights reserved For research use only Not for human or animal therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 48 invitrogen by Lefe technologies Corporate Headquarters 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
63. sed in the Expressway Lumio Cell Free Expression and Detection System e Supercoiled plasmid DNA recommended to obtain the highest yields e Linear DNA e PCR product Many expression vectors or DNA templates may be used For proper expression all templates must contain the T7 promoter an initiation codon and a prokaryotic Shine Dalgarno ribosome binding site RBS upstream of the gene of interest To recombine your gene of interest into pEXP3 DEST or pEXP4 DEST you will need an entry clone containing your gene of interest Many entry vectors are available from Invitrogen to facilitate generation of entry clones For more information go to www invitrogen com or contact Technical Support page 44 Refer to the manual for the specific entry vector you are using for detailed instructions to construct an entry clone pEXP3 DEST supplied with K9900 70 and V960 03 allows you to fuse Lumio and 6xHis tags to the N terminus of your protein of interest using Gateway Technology pEXP4 DEST supplied with K9900 90 and V960 04 allows you to fuse Lumio and 6xHis tags to the C terminus of your protein of interest using Gateway Technology Continued on next page Creating an Expression Clone continued Propagating Vectors Points to Consider Before Recombining into pEXP3 DEST Points to Consider Before Recombining into pEXP4 DEST To propagate and maintain pEXP3 DEST and pEXP4 DEST use 10 ng of the vector to tr
64. ses 1286 1307 Lumio tag bases 1336 1353 Polyhistidine 6xHis region bases 1369 1386 T7 reverse priming site bases 1440 1459 T7 transcription termination region bases 1445 1529 Zeocin resistance gene bases 1673 2047 Ampicillin resistance gene bases 2068 2928 pUC origin bases 3073 3746 Map and Features of pEXP5 NT CALML3 pEXP5 The pEXP5 NT CALML3 vector 3194 bp contains a human calmodulin like 3 NT CALML3 Map gene CALML3 GenBank accession number NM 005185 that has been TOPO Cloned into pEXP5 NT TOPO in frame with the N terminal tag The size of the CALML3 fusion protein is approximately 19 5 kDa The complete sequence of pEXP5 NT CALML3 is available for downloading from www invitrogen com or by contacting Technical Support see page 44 Ep LJ ATG 6xHis TEV CALML3 pEXP5 NT CALML3 3194 bp Comments for pEXP5 NT CALML3 3194 nucleotides T7 promoter bases 1 17 T7 forward priming site bases 1 20 Ribosome binding site RBS bases 68 73 Initiation ATG bases 80 82 Polyhistidine 6xHis region bases 92 109 HisG epitope bases 92 112 TEV recognition site bases 122 142 CALML3 bases 146 595 T7 reverse priming site bases 647 666 T7 transcription terminator bases 608 736 bla promoter bases 848 946 Ampicillin resistance gene bases 947 1807 pUC origin 1952 2625 43 Technical Support Obtaining support Safety Data Sheets SDS Certificate of Analysis Limited warranty 44
65. slyD Extract E coli Reaction Buffer A A and 2X Feed Buffer once or twice is acceptable However avoid multiple freeze thaw cycles as this may result in loss of activity The pEXP3 GW CAT supplied with cat nos K9900 70 V960 03 pEXP4 ORF supplied with cat nos K9900 90 V960 04 or pEXP5 NT CALML3 supplied with cat no K9900 60 control vectors may be used as a positive control for protein expression To propagate and maintain the control plasmid 1 Use the stock solution to transform a recA endA E coli strain like TOP10 DH5a T1 or equivalent Use 10 ng of plasmid for transformation Select transformants on LB agar plates containing 100 ug ml ampicillin Prepare a glycerol stock of a transformant containing plasmid for long term storage Continued on next page 15 General Guidelines for Protein Synthesis continued Choosing a Use the table below to choose the appropriate protocol for your application Protein Synthesis needs Protocol If you wish to Then proceed to Generate Lumio tagged protein and perform real time detection Generate Lumio tagged protein and not perform real time detection Protein Synthesis with Real Time Lumio Detection page 19 Standard Protein Synthesis page 21 16 Protein Synthesis with Real Time Lumio Detection Introduction Real Time Lumio Detection Important Note This section provides information on perf
66. the reagent Protein Synthesis with Real Time Lumio Detection continued Instrument Specifications Performing Protein Synthesis with Real Time Lumio Detection For real time detection of Lumio tagged proteins we recommend performing the protein synthesis reaction in a spectrofluorometer with a built in incubator and mixer e g Molecular Devices Gemini XS Spectrofluorometer The wavelength settings for Lumio detection are Excitation wavelength 500 nm Emission wavelength 535 nm Additional fluorescence spectra information is provided on page 5 If your spectrofluorometer is not equipped with an incubator and or mixer you can incubate the reaction in a thermomixer or shaking incubator water bath and transfer the tubes plates to the spectrofluorometer at regular intervals during the reaction to perform the reading see Alternative next page We do not recommend using a non shaking incubator because it produces a less stable and less consistent temperature environment Use the protocol below to synthesize your protein from the DNA template with real time detection of Lumio tagged proteins 1 Thaw the Lumio Green Detection Reagent and mix well by pipetting up and down 2 Prepare reactions in tubes or microplates suitable for use with your spectrofluorometer For each sample add the following reagents to each tube or well on ice For multiple samples scale up the volume of each reagent accordingly and ali
67. thesis to occur resulting in the achievement of significantly enhanced recombinant protein yields in up to 2 6 hours pos Incubate for 5 5 hours Initiate protein Add Feed Buffer Obtain recombinant protein synthesis 30 minutes after reaction initiating reaction The Expressway Lumio Cell Free Expression and Detection System is suitable for use in the following applications Characterizing proteins Analyzing mutants Verifying cloned gene products Producing proteins that are toxic to cells Real time detection of protein production For more information on the downstream applications of cell free protein expression technologies refer to published reviews Katzen et al 2005 Continued on next page Overview continued Components of the System Features of pEXP3 DEST and pEXP4 DEST Vectors E coli slyD Extract An optimized 30 E coli extract Zubay 1973 for increased stability of DNA constructs during transcription and translation and enhanced signal to background ratio with Lumio detection An optimized feed buffer containing salts and other substrates Kim and Swartz 1999 to replenish components depleted or degraded during protein synthesis thus enhancing recombinant protein yield Proprietary 17 Enzyme Mix containing 17 RNA polymerase and other components optimized for T7 based expression from DNA templates Studier et al 1990 Optimized reaction buffer composed of an ATP r
68. thetic Substitution of Methionine in Proteins by its Analogs 2 Aminohexanoic Acid Selenomethionine Telluromethionine and Ethionine in Escherichia coli Eur J Biochem 230 788 796 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 85 3391 3395 Doublie S 1997 Preparation of Selenomethionyl Proteins for Phase Determination Meth Enzymol 276 523 530 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 Griffin B A Adams S R and Tsien R Y 1998 Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells Science 281 269 272 Hendrickson W A Horton J R and LeMaster D M 1990 Selenomethionyl Proteins Produced for Analysis by Multiwavelength Anomalous Diffraction MAD A Vehicle for Direct Determination of Three Dimensional Structure EMBO J 9 1665 1672 Katzen F Chang G and Kudlicki W 2005 The past present and future of cell free protein synthesis systems Trends Biotechnol 23 150 156 Kim D M Kigawa T Choi C Y and Yokoyama S 1996 A Highly Efficient Cell free Protein Synthesis System from E coli Eur J Biochem 239 881 886 Landy A 1989 Dynamic Structural and Regulatory Aspect
69. universal cloning technology 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 vector systems To express your gene of interest in E coli using the Gateway Technology simply Clone your gene of interest into a Gateway entry vector of choice to create an entry clone Generate an expression clone by performing an LR recombination reaction between the entry clone and pEXP3 DEST or pEXP4 DEST Use your expression clone in the Expressway E coli Expression System with Lumio Technology for in vitro protein synthesis see below For more information about Gateway Technology and performing the LR recombination reaction refer to the Gateway Technology manual available from www invitrogen com or by contacting Technical Support page 44 The table below describes the major steps required to synthesize your recombinant protein of interest using the Expressway Lumio Expression and Detection System Refer to the specified pages for details to perform each step Step Action Pages 1 Generate the DNA template 7 2 Purify your DNA template 13 3 Perform the protein synthesis reaction 14 Optional Perform real time protein synthesis detection 17 4 Analyze tagged recombinant proteins using in gel 26 Lumio detection Lumio Technology Introduction

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