MembranePro Functional Protein Expression System
Contents
1. transfection 293fectin is also available separately see page 30 for ordering information For more information visit www lifetechnologies com or contact Technical Support see page 31 Opti MEM I Reduced Serum Medium is included with the FreeStyle 293 Expression System to facilitate optimal formation of DNA 293fectin complexes Opti MEM lis a modification of Eagle s Minimal Essential Medium buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine thymidine sodium pyruvate L glutamine trace elements and growth factors The protein level is minimal 15 pg mL with insulin and transferrin being the only protein supplements Phenol red is included at a reduced concentration as a pH indicator Opti MEM I Reduced Serum Medium is also available separately see page 29 for ordering information For more information visit www lifetechnologies com or contact Technical Support see page 31 We generally perform transfection experiments in a 30 mL volume To transfect suspension FreeStyle 293 F cells we recommend using the following optimized conditions Final transfection volume 30 mL Number of cells to transfect 3 x 10 cells final cell density of 1 x 10 cells mL Amount of plasmid DNA and MembranePro reagent 9 ug of pEF6 expression construct and 27 uL of MembranePro Reagent Amount of 293fectin 60 pL If you are using other cells you may want to test varying amounts of 293fect2. 31 Product Amount Cat no pEF6 V5 His TOPO TA Expression Kit 20 reactions K9610 20 293FT Cell Line 3 x 107 cells R700 07 Lipofectamine 2000 Transfection Reagent 0 75 mL 11668 027 1 5 mL 1668 019 15 mL 11668 500 Opti MEM I Reduced Serum Medium 100 mL 31985 062 500 mL 31985 070 One Shot TOP10 Chemically Competent E coli 10 reactions C4040 10 20 reactions C4040 03 40 x 50 pL C4040 06 Media Buffers and Werecommend the following media and buffers for culturing passaging Reagents maintaining and transfecting your 293FT cell cultures For more information on these and other cell culture products available from Life Technologies refer to www lifetechnologies com or contact Technical Support see page 31 Note Products are also available in different quantities and packaging sizes Product Amount Cat no Dulbecco s Modified Eagle Medium D MEM 500 mL 11965 092 Dulbecco s Modified Eagle Medium D MEM high 500 mL 11995 065 glucose with L glutamine and sodium pyruvate Opti MEM I Reduced Serum Medium 500 mL 31985 070 Dulbecco s Phosphate Buffered Saline D PBS 1 000 mL 14040 117 1X liquid Phosphate Buffered Saline PBS pH 7 4 1X liquid 500 mL 10010 023 MEM Non Essential Amino Acids Solution 10 mM 100 mL 11140 050 100X liquid MEM Sodium Pyruvate Solution 10 mM 100X 100 mL 11360 070 liquid L Glutamine 200 mM 100X liquid 100 mL 25030 081 Fetal Bovine Serum
3. 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Nguyen D and Hildreth J 2000 Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid enriched membrane lipid rafts J Virology 74 3264 3272 2011 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners For research use only Not intended for any animal or human therapeutic or diagnostic use 33 Headquarters 9 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 o For support visit www invitrogen com support or email techsupport invitrogen com technologies www lifetechnologies com
4. clean and free from contamination with phenol and sodium chloride Isolate the expression construct using the PureLink HiPure Plasmid MaxiPrep kit Ratio of expression construct to MembranePro Reagent is incorrect Co transform 293FT cells with 9 ug of purified pEF6 expression construct and 27 ug of MembranePro Reagent to maintain a 1 3 ratio M MembranePro Precipitation Mix is not mixed well enough The MembranePro Precipitation Mix is slightly viscous To ensure adequate mixing of the MembranePro Precipitation Mix and the culture medium in the collection tube invert the tube gently at least 10 times Do not vortex Cells incubated for too long after the transfections If transfections are performed too early in the afternoon VLPs may be secreted into the growth medium containing the transfection complexes during the night These VLPs will be lost when the medium in changed in the morning For convenience and to maximize particle yield we recommend performing the transfections in the late afternoon after 4 pm with a medium change the following morning before 10 am Serum proteins appear in the VLP precipitate Used non recommended sera in the media Prepare media using the recommended fetal bovine serum FBS see page 29 for ordering information 20 Continued on next page Troubleshooting continued MembranePro Procedures continued Symptom Cause Soluti
5. however the MembranePro Functional Protein Expression System does not support using the tags for extraction and purification BGH reverse priming site Permits sequencing through the insert Bovine growth hormone Efficient transcription termination and BGH polyadenylation polyadenylation of mRNA Goodwin and Rottman signal 1992 fl origin Allows rescue of single stranded DNA SV40 early promoter and Allows efficient high level expression of the origin blasticidin resistance gene and episomal replication in cells expressing the SV40 large T antigen EM 7 promoter For expression of the blasticidin resistance gene in E coli Blasticidin resistance gene Selection of stable transfectants in mammalian cells bsd Kimura et al 1994 SV40 polyadenylation Efficient transcription termination and signal polyadenylation of mRNA pUC origin High copy number replication and growth in E coli bla promoter Allows expression of the ampicillin bla resistance gene Ampicillin resistance gene B lactamase Selection of transformants in E coli 28 Ordering Information Additional Many of the components of the MembranePro Functional Protein Expression MembranePro kits are also available separately from Life Technologies These products are listed below For more information refer to www lifetechnologies com or contact components Technical Support see page
6. vector as a positive control for mammalian transfection and expression The gene encoding galactosidase is expressed in mammalian cells under the control of the human EF 1a promoter A successful transfection will result in P galactosidase expression that can be easily assayed see below However you cannot use this vector as a positive control for VLP formation and secretion because galactosidase is not a membrane protein and it will not be incorporated into the VLPs budding from the 293FT plasma membrane and secreted into the growth medium If you are using a cell line other than 293FT i e a stable cell line we recommend testing your cell line with the pEF6 V5 His TOPO lacZ control or a similar reporter construct to determine its transfectability with Lipofectamine 2000 before attempting VLP formation The lacZ control is provided with the pEF6 V5 His TOPO TA Vector Kit as part of the MembranePro Functional Protein Expression Kit For more information including the map of the vector and a description of its features refer to the pEF6 V5 His TOPO TA Vector Kit manual part no 25 0279 You may assay for P galactosidase expression by activity assay using cell free lysates Miller 1972 or by staining the cells for activity Life Technologies offers the B Gal Assay Kit and the P Gal Staining Kit for fast and easy detection of B galactosidase expression See page 30 for ordering information 15 Harvest Virus Li
7. with VLPs in the presence of MembranePro Precipitation mix e Discontinue using antibiotics at least one passage before transfection e Do not allow cells to grow to confluency e Use cells that have been passaged 3 4 times after the most recent thaw e Use cells that have been subcultured for less than 16 passages e Since 293FT cells are easily dislodged do not squirt transfection complexes directly onto cell monolayer If transfections are performed too early in the afternoon protein production and VLP formation may begin during the night resulting in particle secretion into the growth medium containing the transfection complexes These VLPs will be lost when the medium in changed in the morning For convenience and to maximize particle yield we recommend performing the transfections in the late afternoon after 4 pm with a medium change the following morning before 10 am e MembranePro Reagent e pEF6 expression construct purified according to guidelines on page 11 e 293FT cells at 90 confluency see next page e D MEM complete growth medium supplemented with 0 1 mM MEM Non Essential Amino Acids 1 mM sodium pyruvate and 4 mM L glutamine pre warmed to 37 C e Lipofectamine 2000 transfection reagent mix gently before use e Opti MEM I Reduced Serum Medium e T 175 culture flasks e 15 mL conical tubes e 37 C incubator with a humidified atmosphere of 5 CO Continued on next page 13 Transfect 2
8. 1 Generate the pEF6 expression construct containing your 9 11 gene of interest 2 Co transfect the 293FT producer cell line with your pEF6 12 14 expression construct and the MembranePro Reagent 3 Collect culture medium and clarify it by low speed 15 17 centrifugation Harvest VLPs using the MembranePro Precipitation Mix 17 Resuspend VLPs and proceed to functional assay or aliquot 17 the VLPs and freeze for future use Methods Generate the pEF6 Expression Construct pEF6 V5 His TOPO TA Vector Kit Workflow for Generating the pEF Expression Construct Polymerase Mixtures The MembranePro Functional Protein Expression System is optimized for use with the pEF6 vector pEF6 is a non viral EF 1a promoter driven mammalian expression vector that permits the overexpression of your recombinant protein in a broad range of mammalian cell types Goldman et al 1996 Mizushima and Nagata 1990 For your convenience the pEF6 V5 His TOPO TA Vector Kit is supplied with the MembranePro Functional Protein Expression Kit and it is also available separately from Life Technologies see page 29 The pEF6 V5 His TOPO TA Vector Kit allows you to directly insert a Tag polymerase amplified PCR product into the pEF6 V5 His TOPO vector ina highly efficient 5 minute one step cloning TOPO Cloning reaction to generate your expression vector To clone your gene of interest into the pEF6 V5 His TOPO vector perform th
9. 175 flask yields approximately 500 pL of VLPs following resuspension Depending on the efficiency of expression of the membrane protein of interest a single reaction may generate a VLP sample containing 50 ug to 100 pg of total protein CAUTION When working with mammalian cells handle as potentially biohazardous material under at least Biosafety Level 2 BL 2 containment For more information on BL 2 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 5 ed published by the Centers for Disease Control which is available for downloading at www cdc gov od ohs biosfty bmbl5 bmbl5toc htm CAUTION Although the MembranePro system does not create actual viral particles resultant VLPs may still pose some biohazardous risk if fusogenic particles come in contact with bare skin If used with cells containing or expressing viral genomic sequences you may produce transducing capable VLPs Observe Risk Group 2 RG 2 guidelines for handling and disposing of biohazardous materials For more information on RF 2 guidelines refer to NIH Guidelines for Research Involving Recombinant DNA Molecules which is available for downloading at http oba od nih gov oba rac Guidelines NIH_Guidelines pdf Continued on next page Harvest Virus Like Particles VLPs continued Materials Needed Harvesting Procedure 293FT cells 48 hour post transfection MembranePro Precipitation Mix 50 mL conical centrifuge tube Phosph
10. 500 mL 16000 044 Geneticin Selective Antibiotic liquid 20 mL 10131 027 Continued on next page 29 Ordering Information continued Additional Products The products listed below may be used with MembranePro Functional Protein Expression kits For more information refer to www lifetechnologies com or contact Technical Support see page 31 Product Amount Cat no Platinum Tag DNA Polymerase 100 reactions 10966 018 500 reactions 10966 034 PureLink HiPure Plasmid MaxiPrep kit 10 preps K2100 06 25 preps K2100 07 Countess Automated Cell Counter 1 unit C10227 includes 50 Countess cell counting chamber slides and 2 mL of Trypan Blue Stain Trypan Blue Stain 100 mL 15250 061 B Gal Assay Kit 80 mL K1455 01 P Gal Staining Kit 1 kit K1465 01 Water distilled 20 x 100 mL 15230 196 The products listed below may be used with MembranePro Functional Protein Expression kits for the scalable production of VLPs in FreeStyle 293 F cells For more information refer to www lifetechnologies com or contact Technical FreeStyle 293 Expression System Support see page 31 Product Amount Cat no FreeStyle 293 Expression System 1 kit K9000 01 FreeStyle 293 F cells 1 vial R790 07 1 x 107 cells FreeStyle 293 Expression Medium 1 liter 12338 018 6 x 1 liter 12338 026 293fectin 1mL 12347 019 pCMVeSPORT Bgal 25 ug 10586 014 30 Technical Support Obt
11. 93FT Cells continued Transfection Procedure 14 Day 1 Preparing 293FT Cells for Transfection 1 The day before transfection plate approximately 1 x 107 293FT cells in 25 mL of complete growth medium in a T 175 tissue culture flask see previous page for each transfection Do not include antibiotics in the medium Note If plating a stable cell line other than 293FT for VLP production ensure that the cells are 90 confluent on the day of transfection and that they can be transfected with at least 70 efficiency Incubate cells overnight in a 37 C incubator with a humidified atmosphere of 5 CO Make sure that the cells are 90 confluent on the day of transfection Day 2 Transfection 3 Prepare transfection complexes for each transfection as follows a Ina sterile 15 mL tube combine 9 ug of purified pEF6 expression construct 27 ug of MembranePro Reagent and 4 mL of Opti MEM I Reduced Serum Medium Mix gently Note If you are using a stable cell line other than 293FT combine 36 ug of MembranePro Reagent and 4 mL of Opti MEM I Reduced Serum Medium in a sterile 15 mL tube and mix gently b Ina separate sterile 15 mL tube combine 180 uL of Lipofectamine 2000 mix gently before use and 4 mL of Opti MEM I Reduced Serum Medium Mix the tubes gently and incubate for 5 minutes at room temperature Note Proceed to Step 4 within 25 minutes After incubation combine the diluted DNA Step a with th
12. E Ait invitrogen by Life technologies MembranePro Functional Protein Expression System For capture and display of functional membrane proteins using virus like particles Catalog Numbers A11667 A11668 A11669 and A11670 Revision Date 23 December 2011 Publication Part Number A11610 MAN0001757 technologies Contents Kit Contents and Storage 2 2 eisein ereit ad entint 1 Description ofthe System aaa 5 MembranePre Functional Protein Expression ansiado lei 5 Methods a sr O 9 Generate the pEF6 Expression Construct ueeeesssnenenenssesesnsnsenennnnnnnnennnnnnnsenennsnsnnnnnnnnnnsnnennnnnn 9 Transtect 293ET Cells irna e e aaa Erin ta En 12 Harvest Virtis Like Particle VERS 22 22 ee ee 16 NA AAA A 18 Troubleshooting ii ea 19 App ndix 2a 22 Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells ueee 22 PEF6 VS Fis OPOS Veen aa in 27 Ordering Information eu san e se lek E chee OWS Sahn As 29 Technical SUpport A Re an INNERN a ESE E eao u aooo a Ethe NeT ES 31 Purchaser N tfica HoN NN 32 EE AE E iii is 33 Types of Kits Kit Contents and Storage This manual is supplied with the following products Product Amount Cat no MembranePro Functional Protein Expression Kit 10 reactions A11667 MembranePro Functional Protein Support Kit 10 reactions A11668 60 reactions A11669 600 reactions A11670 Kit Components Each product contains the f
13. PCR product into a pEF6 V5 His TOPO vector is a rapid and efficient process However to ensure proper expression and packaging of your Generating the l et recombinant membrane protein it is important to pay attention to the general expression considerations outlined below Construct e When using the pEF6 V5 His TOPO vector your insert must contain an ATG initiation codon in the context of a Kozak translation initiation sequence for proper initiation of translation in mammalian cells Kozak 1987 Kozak 1990 Kozak 1991 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while having neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG e The pEF6 V5 His TOPO vector contains the V5 epitope and the C terminal polyhistidine 6xHis tag see Note below To express and display a native membrane protein your insert must contain a stop codon For this design your reverse PCR primer to include a stop codon e Donotadd 5 phosphates to your primers for PCR The PCR product synthesized will not ligate into pEF6 V5 His TOPO vector For detailed instructions for cloning your PCR insert and generating a pEF6 expression construct containing your ge
14. aged 3 4 times after the most recent thaw Density of 293FT culture is too high Do not allow cells to grow to confluency Ensure that the monolayer is 90 confluent at time of transfection to avoid cytotoxicity and detachment of cells during manipulation Transfection unsuccessful Follow the transfection protocol exactly as described on pages 12 15 During transfection pay particular attention to the following points e Do not include antibiotics in the medium e Use Opti MEM I Reduced Serum Medium to dilute transfection complexes e Because 293FT cells are easily dislodged do not squirt transfection complexes directly onto cell monolayer Used cells other than 293FT If you are using a cell line other than 293FT i e a stable cell line we recommend testing your cell line with the pEF6 V5 His TOPO lacZ control or a similar reporter construct to determine its transfectability with Lipofectamine 2000 before attempting VLP formation Continued on next page 19 Troubleshooting continued MembranePro The table below lists some potential problems and solutions that help you Procedures troubleshoot the expression and VLP harvest steps in your membrane protein expression experiments Symptom Cause Solution No or little VLPs pEF6 expression Plasmid DNA for transfection into eukaryotic cells recovered but the control transfection worked construct is not pure must be very
15. aining support Safety Data Sheets SDS Certificate of Analysis Limited Warranty 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 sds 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 pr
16. arately see page 29 for ordering information e One Shot TOP10 Chemically Competent E coli for selecting and amplifying the pEF6 expression vector containing your gene of interest for subsequent transfection into 293FT cells Continued on next page MembranePro Functional Protein Expression continued Advantages of MembranePro Functional Protein Expression Possible Applications N e Allows convenient over expression and display of functional membrane proteins including GPCRs Depending on the efficiency of expression of your GPCR and its affinity for your test ligand a single reaction may generate sufficient VLP sample for up to 1000 ligand binding assay data points e Displays membrane proteins on lipoprotein particles released into the growth medium allowing easy harvest e Enriches for receptor specific activity by capturing membrane protein rich lipid rafts e Does not require harsh treatments or detergents which can inactivate membrane proteins e Does not require ultracentrifugation steps for clarifying the medium where strong shear forces can damage the VLPs e Obviates the need to establish stable cell lines for protein expression which carry the risk of down regulation of expression due to protein toxicity TM e Allows the production of VLPs using the scalable FreeStyle 293 Expression System as an alternative see page 22 VLPs produced using the MembranePro Functional Protein Expres
17. ate buffered saline PBS Refrigerated swinging bucket centrifuge e g Sorvall RC3B centrifuge with a HBB 6 rotor 10 11 12 13 Collect the growth medium containing the VLPs from the culture flask by decanting and transfer it to a 50 mL conical centrifuge tube Centrifuge the medium containing the VLPs at 2 000 x g for 10 minutes in a clinical centrifuge with a swinging bucket rotor to pellet the cell debris Carefully transfer clarified sample to a fresh 50 mL conical centrifuge tube using a pipette Do not attempt to transfer the last 2 mL of the sample to avoid transferring any pelletted material Add 1 5 volume of MembranePro Precipitation Mix to the clarified medium i e if harvested medium is 25 mL add 5 mL of MembranePro Precipitation Mix Mix the sample gently by inverting the tube several times until the MembranePro Precipitation Mix is entirely incorporated into the medium Incubate the sample at 4 C overnight at least 18 hours Recover the VLP particles by centrifuging the sample at 5 500 x g for 30 minutes in a refrigerated swinging bucket centrifuge Note Following centrifugation if packaging was successful a yellow white disc or pellet of VLPs should be visible at the bottom of the conical tube see next page Carefully remove the medium by decanting or pipetting Make sure not to dislodge the VLP pellet If complete removal of the culture medium is required for downstream applica
18. cell line that has been adapted to high density serum free suspension growth in FreeStyle 293 Expression Medium The FreeStyle 293 F cells demonstrate high transfection efficiencies with the 293fectin transfection reagent and allow transfections to be performed at large volumes facilitating larger scale VLP production In addition unlike some other serum free media formulations FreeStyle 293 Expression Medium does not inhibit cationic lipid mediated transfection and permits high efficiency transfections without the need to change or add media TM A brief protocol for transfecting FreeStyle 293 F cells is provided below for more information on maintaining and transfecting FreeStyle 293 F cells in suspension culture refer to the FreeStyle 293 Expression System manual part no 25 0439 available at www lifetechnologies com or by contacting Technical Support see page 31 In addition to the MembranePro Functional Protein Expression Kit the following materials are needed for the scalable production of VLPs in FreeStyle 293 F cells See page 30 for ordering information e Suspension FreeStyle 293 F cells cultured in FreeStyle 293 Expression Medium Recommendation Calculate the number of cells that you need for your transfection experiment and expand cells accordingly Make sure that the cells are healthy and greater than 90 viable before proceeding to transfection e MembranePro Reagent e pEF6 expr
19. ctly to cells in culture medium in the presence of serum e You do not have to remove the complexes or change or add medium following transfection however you may remove the complexes 4 6 hours after transfection without loss of activity Note Lipofectamine 2000 is also available separately from Life Technologies see page 29 To facilitate optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium see page 29 Continued on next page Transfect 293FT Cells continued Guidelines for Transfection Materials Needed The health of your 293FT cells at the time of transfection has a critical effect on the success of VLP production Use of unhealthy cells will negatively affect the transfection efficiency resulting in decreased amount of VLP production For optimal VLP production follow the guidelines below to culture 293FT cells before their use in transfection e Ensure that the cells are healthy and greater than 90 viable e Ensure that the monolayer is 90 confluent at time of transfection to avoid cytotoxicity detachment of cells during manipulation and optimal VLP yield e Subculture and maintain cells in complete D MEM medium containing 4 Fetal Bovine Serum FBS 0 1 mM MEM Non Essential Amino Acids 1 mM sodium pyruvate and 4 mM L glutamine Note Using sera other than those recommended in this manual may result in serum proteins co precipitating
20. e steps outlined below and follow the guidelines listed on the next page For detailed instructions on performing these steps refer to the pEF6 V5 His TOPO TA Vector Kit manual part no 25 0279 This manual is supplied as a component of the MembranePro Functional Protein Expression Kit and it is also available for downloading at www lifetechnologies com 1 Generate a PCR product containing your gene of interest with a Tag DNA polymerase e g Platinum Taq DNA Polymerase 2 TOPO Clone your PCR product containing single 3 A overhangs into the pEF6 V5 His TOPO vector and use the reaction to transform One Shot TOP10 Chemically Competent E coli 3 Pick colonies isolate plasmid DNA and screen for insert directionality by sequencing expression clones with primers provided in the kit You may use a polymerase mixture containing Taq polymerase and a proofreading polymerase to produce your PCR product however the mixture must contain a ratio of Taq polymerase proofreading polymerase in excess of 10 1 to ensure the presence of 3 A overhangs on the PCR product If you use polymerase mixtures that do not have enough Tag polymerase or a proofreading polymerase only you may add 3 A overhangs to your PCR product post amplification For more information refer to the pEF6 V5 His TOPO TA Vector Kit manual Continued on next page Generate the pEF6 Expression Construct continued Guidelines for The cloning of a
21. e diluted Lipofectamine 2000 Step b Mix gently Incubate the mixture for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection Note The complexes are stable for 6 hours at room temperature Add the DNA Lipofectamine 2000 complexes Step 5 dropwise to the T 175 tissue culture flask containing the 293FT cells in 25 mL of complete growth medium Mix gently by rocking the plate back and forth taking care not to dislodge the cells Incubate the cells overnight at 37 C in a humidified 5 CO incubator for approximately 18 hours after transfection Day 3 8 The next morning Day 3 remove and discard the medium containing the DNA Lipofectamine 2000 complexes and replace it with 32 mL of complete culture medium without antibiotics Incubate cells for another day at 37 C in a humidified 5 CO incubator The VLPs begin to bud off from the cell membrane and get secreted into the culture medium approximately 48 hours after transfection Note During VLP production 293FT cells may fuse resulting in the appearance of large multinucleated cells known as syncytia This morphological change is normal and does not affect production of the VLPs Continued on next page Transfect 293FT Cells continued Positive Control Assay for B galactosidase Activity You can use the pEF6 V5 His TOPO lacZ expression
22. e in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany Use of this product in conjunction with methods for the introduction of RNA molecules into cells may require licenses to one or more patents or patent applications Users of these products should determine if any licenses are required Blasticidin and the blasticidin resistance gene bsd are for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 email outlicensing lifetech com References Bouamr F Garnier L Rayne F Verma A Rebeyrotte N Cerutti M and Mamoun R 2000 Differential budding efficiencies of human T cell leukemia virus type 1 HTLV 1 gag and gag pro polyproteins from insect and mammalian cells Virology 278 597 609 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Mol Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nuc Acids Res 15 1311 1326 Ciccarone V Chu Y Schifferli K Pichet J P Hawley Nelson P Evans K Roy L and Bennett S 1999 Lipofectamine 2000 Reagent for Rapid Effic
23. eagent supplied with the kit is a proprietary cationic lipid based formulation suitable for the transfection of nucleic acids into eukaryotic cells Lipofectamine 2000 provides the highest transfection efficiency in 293FT cells If you are using a cell line other than 293FT i e a stable cell line we recommend testing your cell line with the lacZ control included in the pEF6 V5 His TOPO TA Vector Kit or a similar reporter construct to determine its transfectability with Lipofectamine 2000 Upon receipt store the Lipofectamine 2000 Reagent at 4 C Do not freeze Continued on next page Kit Contents and Storage continued pEF6 V5 His TOPO pEF6 V5 His TOPO TA Vector Kit included in the MembranePro Functional TA Vector Kit Protein Expression Kit and also available separately see page 29 contains the reagents listed below Upon receipt store the vector kit at 20 C Reagent Composition Amount pEF6 V5 His TOPO vector 10 ng uL plasmid DNA 20 pL 10X PCR Buffer 100 mM Tris HCl pH 8 3 100 pL 500 mM KCI 25 mM MgCl 0 01 gelatin dNTP Mix 12 5 mM dATP 10 uL 12 5 mM dCTP 12 5 mM dGTP 12 5 mM dTTP Salt Solution 1 2 M NaCl 50 pL 0 06 M MgCl T7 Promoter Primer 0 1 ug uL in TE Buffer 20 pL pH 8 0 BGH Reverse Primer 0 1 ug uL in TE Buffer 20 pL pH 8 0 Control PCR Template 0 05 ug uL in TE Buffer 10 uL pH 8 0 Control PCR Primers 0 1 pg pL each in TE Buffer 10 pL pH 8 0 Ster
24. ession construct purified according to guidelines on page 11 e 293fectin transfection reagent store at 4 C until use e Opti MEM I Reduced Serum Medium pre warmed to 37 C TM e FreeStyle 293 Expression Medium pre warmed to 37 C Note Do not add antibiotics to media during transfection because antibiotics may decrease transfection efficiency e 125 mL polycarbonate disposable sterile Erlenmeyer flasks e Orbital shaker in a 37 C incubator with a humidified atmosphere of 8 CO e Room temperature table top centrifuge and sterile conical centrifuge tubes e Reagents to determine viable and total cell counts e Sterile disposable polycarbonate snap cap tubes e Vortex mixer Continued on next page Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells continued 293fectin Opti MEM 1 Reduced Serum Medium Optimal Conditions for 30 mL Transfection 293fectin is a proprietary formulation suitable for transfecting nucleic acids into eukaryotic cells In the FreeStyle 293 Expression System use of 293fectin to transfect FreeStyle 293 F cells provides the following advantages e 293fectin demonstrates high transfection efficiency in suspension FreeStyle 293 F cells cultured in FreeStyle 293 Expression Medium e DNA 293fectin complexes can be added directly to cells in culture medium e Itis not necessary to remove complexes or change or add medium following
25. g and Storage 293FT Cells N Lipofectamine 2000 Upon receipt store each component of the MembranePro Functional Protein Expression System as detailed below Item Shipping Storage MembranePro Precipitation Mix Room Room temperature temperature MembranePro Reagent Wet ice 20 C Lipofectamine 2000 Wet ice 4 C do not freeze 293FT Cells Dry ice Liquid nitrogen pEF6 V5 His TOPO TA Vector Kit Dry ice 20 C One Shot TOP10 Chemically Dry ice 80 C Competent E coli The 293FT Cell Line is supplied as one vial containing 3 x 10 frozen cells in 1 mL of Freezing Medium Note that only the MembranePro Functional Protein Expression Kit includes the 293FT producer cell line Upon receipt store the cells in liquid nitrogen For additional instructions and information on the 293FT cell line see the 293 FT Cell Line manual included in the MembranePro Functional Protein Expression Kit The 293FT Cell Line manual is also available at www lifetechnologies com or by contacting Technical Support see page 31 The MembranePro protocol and media recommendations are optimized for use with 293FT cells for the production of MembranePro particles and they may deviate slightly from the recommendations made in the 293FT cell manual For optimal results follow the media and culture protocol for MembranePro particle production specified in this manual The Lipofectamine 2000 r
26. he cone during resuspension to retrieve all VLPs A B Panels A and B show the images of the 50 mL tube containing the VLPs before and after the centrifugation step step 7 previous page respectively The VLP pellet is clearly visible at the bottom of the tube after centrifugation One T 175 flask yields approximately 500 uL of VLPs following resuspension VLP protein concentration depends on protein expression and purification efficiency and may be between 0 1 ug L and 1 ug uL Depending on the efficiency of expression of your GPCR and the activity of the GPCR for your ligand of interest a single reaction may generate sufficient VLP sample for up to 1000 ligand binding assay data points Troubleshooting Generating the pEF6 Expression Construct Transfection For troubleshooting the problems you may encounter while generating your pEF6 expression construct containing your gene of interest refer to the pEF6 V5 His TOPO TA Vector Kit manual part no 25 0279 The table below lists some potential problems and solutions that help you troubleshoot the transfection step in your membrane protein expression experiments Symptom Cause Solution No VLPs recovered 293FT cells are Ensure that the cells are healthy and greater than 90 and the control unhealthy viable before transfection transfection did not work Use cells that have been subcultured for less than 16 passages Use cells that have been pass
27. hich provides the convenience of a complete kit with all the reagents supplied for 10 reactions and the MembranePro Functional Protein Support Kit which includes the reagents for functional expression of membrane proteins but does not contain the expression vector cloning module or the 293FT host cells The MembranePro Functional Protein Support Kit is offered in three sizes allowing 10 60 or 600 reactions e MembranePro Reagent optimized for high yield packaging and secretion of VLPs that contain the functional membrane protein of interest into the extracellular medium e MembranePro Precipitation Mix for harvesting VLPs released into the growth medium at clinical centrifuge speeds reducing the mechanical damage to the particles by strong shear forces encountered during ultracentrifugation e Lipofectamine 2000 Transfection Reagent for simple high efficiency transfection of the pEF6 expression construct into the host 293FT cells e 293FT cells for high level expression of the recombinant membrane protein as well as the VLP packaging proteins supplied with the MembranePro Functional Protein Expression Kit or available separately see page 29 for ordering information e pEF6 V5 His TOPO TA Vector Kit for generating the pEF6 expression vector containing your gene of interest in a highly efficient 5 minute one step TOPO Cloning reaction supplied with the MembranePro Functional Protein Expression Kit or available sep
28. ient Transfection of Eukaryotic Cells Focus 21 54 55 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Garnier L Ravallec ML Blanchard P Chaabihi H Bossy J P Devauchelle G Jestin A and Cerutti M 1995 Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 gag particles produced in baculovirus infected cells J Virology 69 4060 4068 Goldman L A Cutrone E C Kotenko S V Krause C D and Langer J A 1996 Modifications of Vectors pEF BOS pcDNA1 and pcDNA3 Result in Improved Convenience and Expression BioTechniques 21 1013 1015 Goodwin E C and Rottman F M 1992 The 3 Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation J Biol Chem 267 16330 16334 Kimura M Takatsuki A and Yamaguchi I 1994 Blasticidin S Deaminase Gene from Aspergillus terreus BSD A New Drug Resistance Gene for Transfection of Mammalian Cells Biochim Biophys Acta 1219 653 659 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nuc Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biol 115 887
29. ile Water 1 mL Expression Control Plasmid 0 5 mg mL in TE Buffer 10 pL pEF6 V5 His TOPO lacZ pH 8 0 Continued on next page Kit Contents and Storage continued One Shot TOP10 One Shot TOP10 Chemically Competent E coli included in the MembranePro Chemically Functional Protein Expression Kit are supplied with the following reagents Transformation efficiency of the competent cells is 21 x 10 cfu ng plasmid DNA Competent E coli Store the TOP10 cells at 80 C Reagent Composition Amount S 0 C Medium 2 Tryptone 6 mL 0 5 Yeast Extract 10 mM NaCl 2 5 mM KCl 10 mM MgCl 10 mM MgSO 20 mM glucose TOP10 Chemically Competent E coli 21 x 50 uL pUC19 Control DNA 10 pg pL in 5 mM 50 pL Tris HCI 0 5 mM EDTA pH 8 0 Genotype of TOP10 F mcrA A mrr hsdRMS mcrBC 680lacZAM15 AlacX74 recA1 araD139 A ara leu Cells 7697 galU galK rpsL Str endA1 nupG X Description of the System MembranePro Functional Protein Expression MembranePro Functional Protein Expression Technology How MembranePro Functional Protein Expression Works The MembranePro Functional Protein Expression Technology is a system for the expression and display of mammalian cell surface membrane proteins including G protein coupled receptors GPCRs in an aqueous soluble format The system uses virus like particles VLPs to capture lipid raft regions of the cell s plasma membrane as the VLP
30. in e g 30 40 50 60 80 uL with 30 mL scale transfection 9 uL plasmid DNA 27 uL MembranePro Reagent to determine the optimal transfection conditions A Continued on next page 23 Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells continued Transfection Procedure 24 TM Follow the procedure below to transfect suspension FreeStyle 293 F cells in a TM 30 mL volume Remember that you may keep the cells in FreeStyle 293 Expression Medium during transfection We recommend including a positive control pCMV SPORT Bgal supplied with the FreeStyle 293 Expression System and a negative control no DNA no 293fectin in your experiment 1 The day before transfection day 1 determine the number of cells you need for your experiment For each 30 mL transfection you need 3 x 10 viable cells in 28 mL of FreeStyle 293 Expression Medium Tip To transfect on day 2 seed the cells at a density of 6 x 10 7 x 10 viable cells mL To transfect on day 3 seed the cells at a density of 3 x 10 4 x 10 viable cells mL On the day of transfection transfer a small aliquot of the cell suspension to a microcentrifuge tube and determine cell viability and the amount of cell clumping Vigorously vortex for 45 seconds to break up any cell clumps and determine total cell counts Viability of the cells must be over 90 Important For optimal transfection results make sure that y
31. ited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 27 RNA Transfection Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker 32 The 293FT Cell Line is genetically modified and carries the pUC derived plasmid pCMVSPORT6TAg neo As a condition of sale use of this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms 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 Corporation 5791 Van Allen Way Carlsbad California 92008 This product is licensed from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for us
32. ke Particles VLPs Guidelines for Harvesting VLPs 16 e When harvesting the VLPs from the culture medium 48 hours after the transfection there will likely be floating cell debris Centrifuge the medium briefly to remove the cell debris After centrifugation however do not collect the last 2 mL of the medium to avoid transferring the pelletted debris e You may store the clarified culture medium overnight before isolating the VLPs with the MembranePro Precipitation Mix e The MembranePro Precipitation Mix is slightly viscous To ensure adequate mixing of the MembranePro Precipitation Mix and the culture medium in the collection tube invert the tube gently at least 10 times Do not vortex e Although unnecessary culture medium containing VLPs may be filtered through a 0 45 micron filter to ensure removal of cells However we do not recommend filtration because it reduces the VLP yield e When resuspending the precipitated VLP particles pipet the solution up and down taking care not to introduce air bubbles Do not vortex the solution because it might denature and inactivate your membrane protein of interest e You may store the VLPs for 2 days at 4 C or for up to 6 months at 80 C without any loss of protein activity Before freezing aliquot the VLPs to avoid freezing and thawing the particles more than once Samples which have been subjected to multiple freeze thaw cycles will exhibit reduced activity e One T
33. mized conditions to use when transfecting FreeStyle 293 F cells in a 30 mL volume are listed as a reference Note that transfection conditions vary depending on the type of culture vessel used and the growth conditions of your cells therefore we recommend that you perform pilot studies to optimize your transfection conditions Transfection Amount of Amount of Dilution Amountof 293fectin Lipid DNA Volume Numberof DNA MembranePro Volumet 293fectin Dilution Complex Reagent Volumet Volume 30 mL 3 x 10 9 ug 27 pL to 1 mL 60 pL to 1 mL 2mL 1 liter 300 pg 900 pL to 35 mL 2mL to 35 mL 70 mL Final cell density of 1 x 10 viable cells mL tDiluted in Opti MEM I Reduced Serum Medium A hy The efficiency of the transfection reaction may decrease as the transfection volume increases especially if the FreeStyle 293 F cells are not growing as a single cell suspension i e if there is significant cell clumping Continued on next page 25 Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells continued Harvesting 1 Transfer cultures to 50 mL conical tubes and centrifuge at 1 000 x g for Procedure 10 minutes in a clinical centrifuge with a swinging bucket rotor to pellet and remove the cell debris 2 Transfer the media supernatant to fresh conical tubes and centrifuge at 2 000 x g for 10 minutes to ensure that all cell debris is removed 3 Carefully recover cla
34. ndogenous GPCRs and other receptors are localized in these microdomains after having passed the cellular quality control As the VLP buds from the cell it becomes enveloped in this portion of the plasma membrane and captures the membrane proteins in their native context By capturing just the membrane protein rich lipid rafts this versatile and ready to use system distinguishes itself from crude membrane fractions which contain total plasma membrane as well as contaminating amounts of endoplasmic reticulum Golgi apparatus and nuclear envelope CAUTION Although the MembranePro system does not create actual viral particles resultant VLPs may still pose some biohazardous risk if fusogenic particles come in contact with bare skin If used with cells containing or expressing viral genomic sequences you may produce transducing capable VLPs Observe Risk Group 2 RG 2 guidelines for handling and disposing of biohazardous materials For more information on RF 2 guidelines refer to NIH Guidelines for Research Involving Recombinant DNA Molecules which is available for downloading at http oba od nih gov oba rac Guidelines NIH_Guidelines pdf Continued on next page MembranePro Functional Protein Expression continued Components of MembranePro Functional Protein Expression System The MembranePro Functional Protein Expression System is offered in two configurations The MembranePro Functional Protein Expression Kit w
35. ne of interest refer to the pEF6 V5 His TOPO TA Vector Kit manual part no 25 0279 This manual is supplied as a component of the MembranePro Functional Protein Expression Kit and it is also available for downloading at www lifetechnologies com Y Cloning your gene into the pEF6 V5 His TOPO vector without a stop codon and in frame with the polylinker will result in a fusion protein with V5 and polyhistidine 6xHis tags on the C terminus of your protein As the C terminus of your transmembrane protein will likely be inside the VLP these tags will be inaccessible to purification resins and antibodies In theory these tags could be used to identify and isolate a fusion membrane protein after denaturing the VLP however the MembranePro Functional Protein Expression System does not support using the tags for extraction and purification Continued on next page 10 Generate the pEF6 Expression Construct continued Guidelines for Isolating Plasmid DNA e Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination with phenol and sodium chloride Contaminants may kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency e When isolating plasmid DNA from E coli strains such as TOP10 that are wild type for endonuclease 1 endA1 with commercially available kits ensure that the Lysis or Resuspension Buffer contains 10 mM EDTA EDTA inactiva
36. oducts are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits 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 please 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 31 Purchaser Notification Information for European Customers Limited Use Label License Research Use Only Lim
37. ollowing components For detailed descriptions of the components see pages 24 Component Size Quantity MembranePro Functional Protein Expression Kit 10 reactions MembranePro Reagent 300 ug tube 1 tube MembranePro Precipitation Mix 75 mL bottle 1 bottle Lipofectamine 2000 Transfection Reagent 0 75 mL tube 3 tubes 293FT Cells 3 x 10 cells vial 1 vial pEF6 V5 His TOPO TA Vector Kit 20 reactions kit 1 kit One Shot TOP10 Chemically Competent E coli 20 reactions kit 1 kit MembranePro Functional Protein Support Kit 10 reactions MembranePro Reagent 300 ug tube 1 tube MembranePro Precipitation Mix 75 mL bottle 1 bottle Lipofectamine 2000 Transfection Reagent 0 75 mL tube 3 tubes MembranePro Functional Protein Support Kit 60 reactions MembranePro Reagent 300 ug tube 6 tubes MembranePro Precipitation Mix 75 mL bottle 5 bottles Lipofectamine 2000 Transfection Reagent 15 mL kit 1 kit MembranePro Functional Protein Support Kit 600 reactions MembranePro Reagent 300 pg tube 10 x 6 tubes MembranePro Precipitation Mix 75 mL bottle 10 x 5 bottles Lipofectamine 2000 Transfection Reagent 15 mL kit 10 kits The MembranePro Functional Protein Expression Kit and all its components are for research use only They are not intended for any animal or human therapeutic or diagnostic use Continued on next page Kit Contents and Storage continued Shippin
38. omoter bases 3012 3078 Blasticidin resistance gene bases 3079 3477 V40 early polyadenylation signal bases 3635 3765 pUC origin bases 4148 4821 complementary strand bla promoter bases 21 105 complementary strand Ampicillin b a resistance gene bases 4966 5826 complementary strand Continued on next page 27 pEF6 V5 His TOPO Vector continued Features of pEF6 V5 His TOPO been functionally tested pEF6 V5 His TOPO 5 840 bp contains the following elements All features have Feature Benefit Human elongation factor la hEF 10 promoter Permits overexpression of your recombinant protein in a broad range of mammalian cell types Goldman et al 1996 Mizushima and Nagata 1990 T7 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert TOPO Cloning site Allows insertion of your PCR product in frame with the C terminal V5 epitope and polyhistidine 6xHis tag V5 epitope Allow detection and purification of the fusion Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr C terminal polyhistidine 6x His tag protein however fusion displayed on VLPs normally contain the V5 epitope and the polyhistidine 6x His tag inside the VLP which is inaccessible to purification resins and antibodies In theory these tags could be used to identify and isolate a fusion membrane protein after denaturing the VLP
39. on Membrane proteinis VLPs damaged during When resuspending the precipitated VLP particles not functional or harvest pipet the solution up and down taking care not to shows reduced introduce air bubbles Do not vortex the solution activity because it might denature and inactivate your membrane protein of interest VLPs stored incorrectly before the functional assay You may store the VLPs for 2 days at 4 C or for up to 6 months at 80 C without any loss of protein activity Before storing aliquot the VLPs to avoid freezing and thawing the particles more than once Samples which have been subjected to multiple freeze thaw cycles will exhibit reduced activity Protein of interest is not a membrane protein associated with the plasma membrane lipid rafts You can use the MembranePro Functional Protein Expression System to display most membrane proteins that are destined for the plasma membrane including GPCRs Proteins fated for the Golgi apparatus endoplasmic reticulum or the nuclear envelope cannot be displayed on VLPs 21 Appendix Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells VLP Production in FreeStyle 293 F Cells Materials Needed 22 You can use the FreeStyle 293 Expression System to generate the VLPs in FreeStyle 293 F suspension adapted cells see page 30 for ordering information TM The FreeStyle 293 F cell line is a variant of the 293
40. ou have a single cell suspension It may be necessary to vortex the cells for 10 to 30 seconds Calculate the volume of cell suspension that contains the appropriate number of cells for one transfection for each 30 mL transfection you need 3 x 10 viable cells Place the shaker flask containing cells in a 37 C incubator on an orbital shaker Prepare lipid DNA complexes for each transfection as follows a Inasterile 15 mL tube dilute 9 ug of purified pEF6 expression construct and 27 ug of MembranePro Reagent 27 uL of the supplied 1 pg pL solution in Opti MEM I Reduced Serum Medium to a total volume of 1 mL Mix gently b Ina separate sterile 15 mL tube dilute 60 uL of 293fectin mix gently before use in Opti MEM I Reduced Serum Medium to a total volume of 1 mL Mix the tubes gently and incubate for 5 minutes at room temperature Note Longer incubation times may result in decreased transfection efficiency After incubation combine the diluted DNA Step a with the diluted 293fectin Step b to obtain a total volume of 2 mL Mix gently Incubate the mixture for 20 30 minutes at room temperature to allow the DNA 293fectin complexes to form While the DNA 293fectin complexes are incubating remove the cell suspension from the incubator and transfer the appropriate volume see step 3 into each sterile disposable 125 mL Erlenmeyer shaker flask Add fresh pre warmed FreeStyle 293 Expression Medium up to a to
41. rified supernatant using a 10 mL pipette leaving the last 1 2 mL of media in the tube to avoid a carry over of cell debris 4 Recover the VLPs using MembranePro Precipitation mix as described in the standard MembranePro protocol see page 17 26 pEF6 V5 His TOPO Vector Map of pEF6 V5 His TOPO The figure below summarizes the features of the pEF6 V5 His TOPO vector The vector is supplied linearized between base pairs 1 760 and 1 761 This is the TOPO Cloning site Unique restriction sites flanking the TOPO Cloning site are shown The pEF6 V5 His TOPO vector is supplied with the MembranePro Functional Protein Expression Kit and it is also available separately from Life Technologies see page 29 For more information on the pEF6 V5 His TOPO vector refer to the pEF6 V5 His TOPO TA Vector Kit manual part no 25 0279 The complete sequence for pEF6 V5 His TOPO is available for downloading at www lifetechnologies com or by contacting Technical Support see page 31 A A PCR Product Comments for pEF6 V5 His TOPO 5840 nucleotides EF 1a promoter bases 470 1653 T7 promoter priming site bases 1670 1689 TOPO Cloning site bases 1760 1761 V5 epitope bases 1826 1867 Polyhistidine 6xHis tag bases 1877 1894 BGH reverse priming site bases 1917 1934 BGH polyadenylation signal bases 1923 2147 f1 origin of replication bases 2193 2621 SV40 promoter and origin bases 2626 2970 EM 7 pr
42. s are secreted from the cell Using this system it is possible to capture and display endogenous or overexpressed GPCRs and other cell surface membrane proteins in their native context for downstream assays Because the VLPs are packaged by the cell and secreted into the culture medium VLPs allow the isolation of functional membrane proteins by simply decanting and clarifying the culture medium and isolating the VLPs by precipitation This represents a substantial savings in time effort and required machinery over preparing cell membrane fractions Because VLPs capture receptor rich regions of the plasma membrane your GPCR may also be substantially enriched over crude membrane preparations Virus like particles VLPs are subviral particles that self assemble from virus derived core structural proteins HIV 1 gag pol rev and VSV G Because VLPs lack a viral genome none of the structural genes are packaged into MembranePro particles As the system does not provide a nucleic acid payload the resultant particles are non transducing non infectious and non replicating The MembranePro Functional Protein Expression System takes advantage of the functionality of the lentiviral gag protein which when expressed in 293FT cells travels to the plasma membrane where it forms buds underneath the lipid rafts Because lipid rafts play an active role in regulating the conformational state and dynamic sorting of membrane proteins recombinant and e
43. sion Kits can substitute for membrane fractions in a variety of downstream applications These may include ligand binding experiments or other functional or biochemical assays Note There are many factors that affect protein yield solubility and function Therefore your expressed membrane protein might not be suitable for all the downstream applications listed above TM You can use the MembranePro Functional Protein Expression System to express package into and display on VLPs most membrane proteins that are destined for trafficking to the plasma membrane including GPCRs Proteins fated for the Golgi apparatus endoplasmic reticulum or the nuclear envelope cannot be displayed on VLPs Efficiency of capture of your protein by VLPs depend on the level of protein expression and the localization of your protein on or near lipid rafts As an alternative to the 293FT cells provided with the MembranePro Functional Protein Expression Kit you can also use the scalable FreeStyle 293 Expression System to generate VLPs see page 22 for the alternative protocol Continued on next page MembranePro Functional Protein Expression continued Experimental The table below describes the major steps required to synthesize your recombinant Outline membrane protein of interest using the MembranePro Functional Protein Expression Kit Refer to the specified pages for details to perform each step Step Action Page
44. tal volume of 28 mL for a 30 mL transfection Continued on next page Scalable Production of Virus Like Particles VLPs in FreeStyle 293 F Cells continued Transfection Procedure continued Optimizing VLP Production Scaling Up Transfections 8 After the DNA 293fectin complex incubation is complete add 2 mL of the complex into each shaker flask from Step 7 To the negative control flask add 2 mL of Opti MEMP I instead of the DNA 293fectin complex Each flask should contain a total volume of 30 mL with a final cell density of approximately 1 x 10 viable cells mL 9 Incubate the cells in a 37 C incubator with a humidified atmosphere of 8 CO in air on an orbital shaker rotating at 125 rpm 10 Harvest the cells or media if recombinant protein is secreted at approximately 48 hours post transfection and assay for recombinant protein expression Expression levels may vary depending on the nature of your membrane protein therefore you may want to perform a time course i e harvest cells or media at 24 48 72 and 96 hours post transfection to optimize expression and capture of your protein It is possible to perform transfections in a larger e g 1 liter volume To transfect suspension FreeStyle 293 F cells in a larger volume scale up the volume of each reagent accordingly The table below lists suggested conditions to use when transfecting FreeStyle 293 F cells in a 1 liter volume The opti
45. tes the endonuclease and avoids DNA nicking and vector degradation e Resuspend the purified plasmid DNA in sterile water or TE Buffer pH 8 0 toa final concentration ranging from 0 1 3 0 ug mL You will need 9 ug of the expression plasmid for each transfection e To ensure that the plasmid DNA used for transfection is sterile you may filter sterilize it through a 0 22 um filter before use IMPORTANT Do not use mini prep plasmid DNA for 293FT transfection We recommend preparing pEF6 plasmid DNA using the PureLink HiPure Plasmid MaxiPrep kit which contains 10 mM EDTA in the Resuspension Buffer see page 30 for ordering information 11 Transfect 293FT Cells 293FT Cell Line N Transfection Methods Lipofectamine 2000 Opti MEM 12 CAUTION When working with mammalian cells handle as potentially biohazardous material under at least Biosafety Level 2 BL 2 containment For more information on BL 2 guidelines refer to Biosafety in Microbiological and Biomedical Laboratories 5 ed published by the Centers for Disease Control which is available for downloading at www cdc gov od ohs biosfty bmbl5 bmbl5toc htm The human 293FT Cell Line is supplied with the MembranePro Functional Protein Expression Kit to facilitate optimal viral like particle VLP production For more information on culturing and maintaining 293FT cells refer to the 293FT Cell Line manual part no 25 0504 This manual supplied
46. tions e g for protein determination on VLPs rinse the centrifuge tube with MembranePro Precipitation Mix diluted 1 5 in 1X PBS Otherwise proceed to step 12 Centrifuge the sample again for 5 minutes at 5 500 x g for 5 minutes in a swinging bucket centrifuge Carefully remove the supernatant by pipetting Resuspend the VLP pellet in 500 uL of PBS or the desired amount of assay buffer by repeatedly pipetting it up and down Be careful not to create froth in the sample Do not vortex The resuspended sample will appear slightly turbid Note Any particulate matter that cannot be resuspended by repeated pipetting or by smearing against the tube wall using the pipettor tip can be left in suspension or separated from the sample by allowing it to settle to the bottom of the tube Proceed to the desired assay You may also store the VLPs at 4 C for 2 days or at 80 C for up to 6 months without any loss of activity if the samples are not subjected to repeated freeze thaw cycles 17 Expected Results Successful VLP Packaging Expected Yield 18 If VLP packaging was successful a yellow white pellet of VLPs should be visible at the bottom of the conical tube following centrifugation step 7 previous page This pellet may not be as distinct as in the pictures below however when the VLP pellet is resuspended in PBS Step 12 previous page the resulting solution will appear slightly cloudy Be sure to rinse the walls of t
47. with the MembranePro Functional Protein Expression Kit is also available at www lifetechnologies com or by contacting Technical Support see page 31 Note The 293FT Cell Line is also available separately from Life Technologies page 29 The MembranePro protocol and media recommendations are optimized for use with 293FT cells for the production of VLPs and they may deviate slightly from the recommendations made in the 293FT cell manual For optimal results follow the media and culture protocol for VLP production specified in this manual The 293FT Cell Line is generally amenable to transfection using standard methods including calcium phosphate precipitation Chen amp Okayama 1987 Wigler et al 1977 lipid mediated transfection Felgner et al 1989 Felgner amp Ringold 1989 and electroporation Chu et al 1987 Shigekawa amp Dower 1988 We typically use cationic lipid based transfection reagents to transfect 293FT cells and recommend the Lipofectamine 2000 transfection reagent for best results The Lipofectamine 2000 reagent supplied with the MembranePro kits Ciccarone et al 1999 is a proprietary cationic lipid based formulation suitable for the transfection of nucleic acids into eukaryotic cells Using Lipofectamine 2000 to transfect 293FT cells offers the following advantages e Provides the highest transfection efficiency in 293FT cells e You can add the DNA Lipofectamine 2000 complexes dire
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