BacPAK™ Baculovirus Expression System User Manual
Contents
1. 6 10 11 12 13 Notes e Transfecting the cells with plasmid DNA alone provides a control that will reveal any contamination in the reagents Baculovirus DNA is large and easily damaged by shearing and BacPAK6 DNA will lose its infectivity if it is damaged Therefore the viral DNA should be handled with care throughout these procedures for example to mix solutions containing BacPAK6 DNA gently flick the tube rather than vortexing it Add 4 ul of the Bacfectin to the DNAs and mix gently Incubate at room temperature for 15 min to allow the transfection reagent to form complexes with the DNA Meanwhile remove the medium from the cell monolayers and add 1 5 ml of BacPAK Grace s Basic Medium Add the Bacfectin DNA mixture dropwise to the medium while gently swirling the dish to mix Incubate at 27 C for 5 hr lf positive control was omitted add 1 5 ml of BacPAK Complete Medium to experimental and negative control dishes Incubate at 27 C If positive control was included add 48 ul of X gluc 25 mg ml in DMSO to 4 ml of BacPAK Complete Medium final concentration 300 ug ml Add 1 5 ml of BacPAK Complete Medium X gluc to the negative and positive control dishes Add 1 5 ml of BacPAK Complete Medium to the experimental dish Incubate at 27 C Blue color will be visible in the positive control dish 5 days after adding the Bacfectin DNA mixture The color indicates successful cotransfection and ge2. Seguences in and around the pBacPAK8 multiple cloning sites Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 31 BacPAK Baculovirus Expression System User Manual Appendix A Vector Maps and MCS Seguences continued AlwN I BstX I MCS 4950 314 BamH 1 1253 Mlul Xma 546 Smal EcoR I EcH36 ll Sacl Asp718l Amp pBacPAK9 Pooiyhedtin pel g 5 5 kb si SnaBl Xha signal SY BstB Hind PO i Mam Su Sse8387 I Eagl Dra lll 3075 Noti Pac 10325 1250 bp AcMNPV Scal 3990 1433 bp M13 ori AcNPV Figure 5 Map of pBacPAK9 Transfer Vector me Bac1 Primer TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA EcoRV 1201 1 e_ polyhedrin promoter AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA 1251 e CGGATCCCGG GAATTCGAGC TCGGTACCAG ATCTTCTAGA TTCGAACTCG BamHI EcoRI Ecl136ll Asp718l Bglll Xbal BstBl Xhol Xmal Sacl Kpnl 1301 Smal stops e i AGGCCTGCAG GGCGGCCGCT TAATTAATTG ATCCGGGTTA TTAGTACATT Stul Eagl Pacl Sse83871 NOt 1351 Pstl TATTAAGCGC TAGATTCTGT GCGTTGTTGA TTTACAGACA ATTGTTGTAC GCG ATCTAAGACA CGCAAC SnaBl a SO 1401 Bac2 Primer e GTATTTTAAT AATTCATTAA ATTTATAATC Figure 6 Sequences in and around the pBacPAK9 multiple cloning sites Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression Syst
3. dry off the containers and flame the necks before mixing the agarose and medium Gently add 1 5 ml of the agarose overlay to each dish Note Allow the agarose to run down the side of the dish taking care not to disturb the cells When the agarose overlay has set add 1 5 ml of BacPAK Complete Medium to each dish Place dishes in a plastic storage box with a moist paper towel as described in the note to Step 2 incubate dishes at 27 C for 7 days Note Stain for virus plaques half the dishes will be stained with neutral red only and half with neutral red and X gal Dilute neutral red to 0 03 with PBS 1 ml of 0 33 w v neutral red stock 10 ml of PBS Add 1 ml of the 0 03 neutral red solution to each of the 10 dishes Incubate at 27 C for 2 3 hr Aspirate off the stain invert the dishes and leave them in the dark at room temperature overnight to allow the plaques to clear and the blue color to fully develop Notes e Neutral red is taken up by healthy cells but not by dead cells Therefore on the dishes stained with neutral red only plaques will be clear circles about 0 5 3 mm in diameter against a red or pink background On the dishes stained with X gal and neutral red plaques will be blue against a red background You should see blue foci in these dishes even if the plaques are small Practice the plaque assay until you can see plaques with neutral red stain alone Neutral red is light sensitive and will become gra
4. 4 dishes each with the 107 10 and 107 dilutions of the cotransfection supernatant gently add 100 ul of the appropriate virus inoculum to the center of the dish take care not to displace any cells For controls infect one dish with 100 ul of the appropriate BacPAK6 virus dilution and place 100 ul of the dilution medium on the remaining dish These controls will be useful for comparison with the infected dishes the negative control will reveal contamination in the reagents Incubate at room temperature for 1 hr on a level surface to allow the virus to infect the cells During this incubation melt 12 ml of 2 SeaPlaque agarose in H O previously autoclaved and cool to 37 C Prewarm 12 ml of BacPAK Complete Medium to 37 C Clontech Laboratories Inc www clontech com Protocol No PT1260 1 2 Version No PR862558 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued 10 11 12 13 14 15 16 17 Note To assay for recombinant viruses in the control cotransfection use BacPAK Complete Medium containing 300 ug ml X Gluc Final concentration in agarose overlay is 150 ug ml Remove the virus inoculum from the cells by tilting the dish and aspirating from the edge Add warm BacPAK Complete Medium to the agarose and mix this makes a 1 agarose solution which is used to overlay the infected cell monolayer to prevent virus progeny from
5. color from the chromogenic GUS substrate X Gluc 1 Remove an aliquot of exponentially growing Sf21 cells and dilute with prewarmed 27 C BacPAK Complete Medium to make a 6 ml cell suspension at a concentration of 7 x 10 cells ml 2 Add 1 5 ml of cell suspension approximately 1 x 109 cells to 2 or 3 35 mm tissue culture dishes and rock to distribute the cells Place in a plastic storage box with a moist paper towel and incubate at 27 C for 1 2 hr 3 Remove the medium from the cells and add 2 ml of BacPAK Grace s Basic Medium Swirl gently remove the medium again and add 2 ml of BacPAK Grace s Basic Medium Incubate at room temperature for 10 30 min while the Bacfectin DNA mixture is prepared as described in the following steps Note A component in serum inhibits transfection this washing step is necessary to replace normal medium with serum free medium before adding the Bacfectin DNA mixture to the cells 4 Dilute the plasmid DNA to 100 ng ul with TE buffer 5 Make the following additions to two or three sterile microfuge tubes optional Tube 1 Tube 2 Tube 3 Experiment Control Control Sterile H O 86 ul 91 ul 86 ul Plasmid DNA 100 ng ul 5 ul 5 ul pBacPAK8 GUS 100 ng ul 5 ul BacPAK6 viral DNA 5 ul 5 ul Bsu36 I digest Final Volume 96 ul 96 ul 96 ul Version No PR862558 19 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued
6. inoculum overlay before adding the 196 agarose overlay Cell monolayer Cells may have been disturbed Avoid touching the cell layer contains holes by addition of virus inoculum with pipettes and tips or the agarose overlay and make additions gently Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual VII Construction of a Recombinant Transfer Vector A Tailoring the Insert If using the vectors provided in the kit the target gene must have its own ATG initiation codon which should be the first ATG in the insert The coding sequence must end with a translation termination codon If you want the target protein to be secreted or directed to the membrane the inserted gene should have the appropriate signal peptide and hydrophobic anchorage encoding sequences Many mammalian signals are recognized in insect cells The target gene should not contain introns use cDNA The 5 untranslated leader sequence should be as short as possible remove leader regions with a high GC content or stable secondary structures if possible Transcription of the inserted gene is terminated by the polyhedrin polyadenylation signal in the transfer vector B Inserting the Target Gene into the Transfer Vector 1 2 Clone the insert into the appropriate site of pBacPAK8 or pBacPAK9 or other suitable vector Screen fortransfe
7. spreading to other areas of the dish Note Water baths are a major source of microbial contamination therefore dry off the containers and flame the necks before mixing the agarose and medium Gently add 1 5 ml of the agarose overlay to each dish Note Allow the agarose to run down the side of the dish taking care not to disturb the cells When the agarose overlay has set add 1 5 ml of BacPAK Complete Medium to each dish Note To assay for recombinant viruses in the control cotransfection use BacPAK Complete Medium containing 150 ug ml X gluc Place dishes in a plastic storage box with a moist paper towel incubate dishes at 27 C for 7 days Dilute neutral red to 0 03 with PBS 1 5 ml of 0 33 w v neutral red stock 15 ml of PBS Add 1 ml of the 0 03 neutral red solution to each of the 14 dishes Incubate at 27 C for 2 3 hr Aspirate off the stain invert the dishes and leave them in the dark at room temperature overnight to allow the plaques to clear Inspect dishes for viral plaques Find dishes on which the diluted cotransfection supernatant produced only a few plaques and mark well isolated ones by circling them with a pen on the outside bottom of the dish Examine under a microscope to ensure that they contain virus infected unstained cells and are not just holes in the cell monolayer Positive control cotransfection In the presence of X gluc recombinant viruses expressing GUS will give rise to blue plaques that
8. vary in size Small underdeveloped plaques may appear clear but will often become blue on longer incubation Prepare a sterile microcentrifuge tube containing 0 5 ml of BacPAK Complete Medium for each well isolated plaque that you have identified Pick the marked plaques by pushing the tip of a sterile Pasteur pipette through the agarose overlay into the plaque and gently sucking a plug of agarose into the pipette tip Wash the agarose plug into the microcentrifuge tube Vortex and store at 4 C overnight to Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued allow viruses to diffuse out of the agarose This is called a plague pick Note Nearly all plaques produced from a BacPAK6 cotransfection are recombinant However we recommend picking up to 10 well isolated plaques Test three to four of these putative recombinant viruses for the target gene and store the remainder at 4 C Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual IX Virus Propagation and Evaluation A Preparation of Passage One Virus Stock Use plague picks to generate virus infected cell proteins or DNA This step also amplifies the virus in the plague pick Seed 35 mm dishes one for each plaque pick w
9. virus contaminated materials including fluids must be autoclaved or disinfected with 5 bleach or a chemical disinfectant before disposal B Culture Media You may propagate Sf21 cells in BacPAK Complete Medium Cat No 631403 which is fully supplemented You may also use serum freemediasuchas BacPAK Grace sBasicMedium Cat No 631404 for assaying or purifying secreted proteins Insectcell mediumdoesnotcontain pH indicators and is pale yellow The pH of the medium is about 6 2 and it will gradually rise as the cells grow however pH will usually not exceed 6 4 BacPAK Complete Medium contains TNM FH medium Grace s Basic Medium Grace 1962 with yeastolate lactalbumin hydrolysate and L glutamine Hink 1970 supplemented with 10 FBS and 50 ug ml gentamycin You may substitute fully supplemented BacPAK Grace s Basic Medium Cat No 631404 for BacPAK Complete Medium throughout these protocols Alternatively prepare TNM FH medium Hink 1970 and supplement as follows 1 Add 50 ml of fetal bovine serum cell culture grade preferably insect cell tested to a 500 ml bottle of TNM FH medium to give 10 v v FBS Clontech Laboratories Inc www clontech com Protocol No PT1260 1 10 Version No PR862558 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued 2 If desired add antibiotics e g 50 units of penicillin and 50 ug of streptomycin per ml of medium or 50 ug of gent
10. 0 ul of the virus inoculum to the center of the dish taking care not to displace any cells Infect 4 dishes with the 109 and 4 with the 10 dilution of BacPAK6 Plate 100 ul of the dilution medium onto the remaining two dishes These dishes will be useful for comparing with the infected dishes and they provide a control that will reveal any contamination in the reagents Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual VI Plague Assay Method continued 6 7 10 11 12 13 14 15 Incubate at room temperature for 1 hr on a level surface to allow the virus to infect the cells During this incubation melt 10 ml of 2 agarose 2 SeaPlaque agarose in H O previously autoclaved and cool to 37 C Prewarm 10 ml of BacPAK Complete Medium to 37 C Add 24 ul of X gal 25 mg ml in DMF per ml to the prewarmed BacPAK Complete medium 240 ul 10 ml or final concentration 12 ul ml medium when mixed with 2 agarose Remove the virus inoculum from the cells by tilting the dish and aspirating from the edge Proceed immediately to step 10 Add warm BacPAK Complete Medium to the agarose and mix this makes a 1 agarose solution which is used to overlay the infected cell monolayer to prevent virus progeny from spreading to other areas of the dish Note Water baths are a major source of microbial contamination therefore
11. 0 ul of virus dilution per plate Plate out an appropriate dilution of BacPAK6 virus and 100 ul of medium as controls Follow the procedure in Section VI A modifying volumes to match the total number of dishes being used After the appropriate incubation time count the plaques on the plates with reasonable numbers of plaques and calculate the virus titer as explained in Section VI B Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual X Characterizing Recombinant Gene Expression Before producing the target protein on a large scale characterize gene expression from the recombinant virus and determine the time course of protein production You can infect cell monolayers of various sizes Table ll to obtain protein or RNA for analysis Always include an infection with the parental BacPAK6 virus which expresses B galactosidase to high levels and a mock infected control With these controls you can find protein or RNA present in recombinant virus infected cells but not in uninfected cells or in cells infected with wild type or parental viruses TABLE II GUIDELINES FOR PREPARING CELLS FOR ANALYSIS OF TARGET GENE PRODUCTION Number of cells to be seeded Virus Volume Dish flask 2hr overnight Inoculum of Medium 35 mm dish 1 5 1 0 x 108 0 1 0 5 ml 1 5 2 0 ml 60 mm dish 2 5 2 0 x 108 0 4 1 0 ml 3 0 5 0 ml 150 mm dish 15 0 10 0 x 106 2 0 6 0 m
12. 260 1 26 Version No PR862558 BacPAK Baculovirus Expression System User Manual IX Virus Propagation and Evaluation continued 3 Incubate at 27 C for 4 6 days until the cells are well infected 4 Centrifuge infected cells at 1 000 x g for 5 min to remove cells and debris 5 Transfer the supernatant to a fresh sterile tube This is the Passage Two virus stock Freeze 5 x 5 ml aliquots at 70 C for long term storage Store the remainder at 4 C and use as the current working stock 6 Determine the titer of the Passage Two stock Section IX E The titer should be gt 5 x 107 pfu ml 7 When the current working stock is depleted thaw an aliquot of Passage Two stock and generate a new working stock by infecting a 50 200 ml suspension culture Steps 1 6 above Note Do not passage the virus repeatedly baculoviruses can accumulate mutations E Titration of Amplified Virus Stocks You must obtain an accurate titer for a virus stock so that you can optimize subsequent infections to produce maximal yield of recombinant protein or the highest titer of virus The quickest and easiest method for determining titer is to use the BacPAK Baculovirus Rapid Titer Kit Cat No 631406 With this kit you can obtain a titer in as little as 48 hours Alternatively you may perform a plaque assay Section VI A In this case dilute amplified virus stock to 104 10 and 10 and infect 2 or 3 dishes of cells for each dilution Use 10
13. Complete Medium DMSO to the cell suspension and mix Keep on ice Place 1 ml aliguots of cells into each vial and cap tightly Ifavailable placevialsin a vapor phase chamber ofthe liquid nitrogen container and freeze cells slowly overnight before placing in the liquid phase Alternatively place vials at 20 C for 1 2 hrandthenina 70 C freezer overnight Transfer to liquid nitrogen as rapidly as possible After a week or two retrieve one vial and test the viability of the stored cells by following the protocol in Section V C Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual VI Plague Assay Method Plague assays are designed to produce distinct viral plagues in a monolayer of host cells under conditions where each plague is the result of a cell being infected by a single virus Plague assays can thus be used to isolate an individual recombinant virus from the pool of viruses generated by a cotransfection SectionVIll C Plaque assays can also be used to determine the titer of a virus stock however titers can be obtained more quickly and easily using Clontech s BacPAK Baculovirus Rapid Titer Kit Cat No 631406 If plaque assays are to be used either to produce a pure recombinant virus clone or to titer virus stocks it is advisable to develop good plaque assay technique by practicing using the virus stocks provided A Pr
14. D Amplification of Recombinant Viruses Preparing Passage Two Virus Stock 26 E Titration of Amplified Virus Stocks 27 X Characterizing Recombinant Gene Expression 28 XI Large scale Target Protein Production 29 Clontech Laboratories Inc www clontechcom Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual Table of Contents continued XII References 30 Appendix A Vector Maps amp Multiple Cloning Site Sequences 31 List of Figures Figure 1 Target gene transfer to baculovirus expression vector 5 Figure 2 Schematic diagram outlining BacPAK procedure 9 Figure 3 Map of pBacPAK8 Transfer Vector 31 Figure 4 Seguences in and around the pBacPAK8 MCS 31 Figure 5 Map of pBacPAK9 Transfer Vector 32 Figure 6 Seguences in and around the pBacPAK9 MCS 32 Figure 7 Map of pBacPAK8 GUS Transfer Vector 33 List of Tables Table I Guidelines for seeding densities 13 Table ll Guidelines for preparing cells for analysis of gene production 28 Protocol No PT1260 1 www clontech com Clontech Laboratories Inc 3 Version No PR862558 BacPAK Baculovirus Expression System User Manual l Introduction Baculovirus gene expression is a popular method for producing large quantities of recombinant proteins in insect host cells In most cases posttranslational processing of eukaryotic proteins expressed in insect cells is similar to protein processing in mammalian cells As a resul
15. M sodium acetate and 1 ml of ethanol Chill at 20 C for 10 min Pellet the DNA in a microcentrifuge at room temperature for 5 min Add 0 5 ml of 75 ethanol vortex briefly and then repellet the DNA Repeat the ethanol wash Dry the pellet at room temperature for 30 min Add 50 ul of TE buffer to pellet and soak at 4 C overnight Gently resuspend DNA using a pipette tip You may digest aliquots of this DNA with a restriction enzyme and analyze by Southern blotting C Processing and Storage of the Passage One Virus Stock 1 After confirming that the plaque comrpises recombinant virus containing the target gene transfer 1 ml of recombinant virus Passage One stock to 70 C for long term storage Cryogenic agents are not required 2 Determine the titer of the Passage One stock of recombinant virus Section IX E You may use the BacPAK Baculovirus RapidTiter Kit Cat No 631406 or a plaque assay The titer should be in the range 1 5 x 107 pfu ml D Amplifying Recombinant Viruses Preparing Passage Two Virus Stock 1 Seed a 50 ml suspension culture with 1 x 10 cells ml and incubate at 27 C until the cell density reaches 4 5 x 10 cells ml 2 days 2 Calculate the volume of the Passage One virus stock that will contain 0 1 pfu for every cell in the suspension culture Multiplicity of Infection M O I 0 1 and add this volume to the suspension culture Clontech Laboratories Inc www clontech com Protocol No PT1
16. T S Z G Bm o UU BacPAK Baculovirus Expression System User Manual Clontech Cat No 631402 oe PT1 260 1 PR862558 Asia Pacific Published 5 June 2008 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Clontech Laboratories Inc ATakara Bio Company 1290 Terra Bella Ave Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com BacPAK Baculovirus Expression System User Manual Table of Contents l Introduction 4 Il List of Components 6 Ill Additional Materials Required 7 IV Experimental Outline 8 V Insect Cell Culture Guidelines 10 A General Considerations 10 B Culture Media 10 C Establishing the Sf21 Cell Line 11 D Subculturing Sf21 Monolayers 11 E Suspension Cultures of Sf21 Cells 13 F Storing Insect Cells in Liquid Nitrogen 14 VI Plaque Assay Method 15 A Practice Plaque Assay 15 B Calculation of Virus Titer 17 C Troubleshooting Plaque Assays 17 VII Construction of a Recombinant Transfer Vector 18 A Tailoring the Insert 18 B Inserting the Target Gene into the Transfer Vector 18 VIII Construction of a Recombinant Viral Expression Vector 19 A Generating a Recombinant Virus 19 B Troubleshooting Guide 21 C Isolating Recombinant Viruses 22 IX Virus Propagation and Evaluation 25 A Preparation of Passage One Virus Stock 25 B Evaluation of Recombinant Viruses 25 C Processing and Storage of the Passage One Virus Stock 26
17. actice Plaque Assay BacPAK6 is a convenient virus for practice because it expresses B galactosidase Kitts amp Possee 1993 and produces plaques that can be stained blue with X gal 1 Remove an aliquot of exponentially growing Sf21 cells that have a viability of gt 95 and dilute with prewarmed BacPAK Complete Medium to make 18 ml of cell suspension at 7 x 10 cells ml 2 Add 1 5 ml of the cell suspension to a 35 mm tissue culture dish and rock to distribute evenly Repeat for 9 more dishes Each dish will receive approximately 1 x 10 cells Incubate the cultures at 27 C for 1 4 hr Notes The correct cell density is critical to assay success e To minimize problems with medium evaporation from the culture dishes during the incubation period place the dishes in a plastic storage box that has a tight fitting lid place a folded moist paper towel inside the box next to the dishes Seeding dishes with a volume of cell suspension less than 1 5 ml will result in an uneven distribution of cells over the dish The volume added to each dish should be between 1 5 and 2 5 ml 3 Make serial 1 10 dilutions of the BacPAK6 Virus Stock provided in the kit in BacPAK Complete Medium to give final dilutions of 10 and 10 4 Inspect the dishes to ensure that the cells have attached to form an even monolayer of about 70 80 confluency Aspirate the medium from the cells using a sterile pasteur pipette or pipette tip 5 Gently add 10
18. amycin per ml of medium from a filter sterilized concentrated stock solution Note Antibiotic use is optional butstronglyrecommendedforcotransfections plaque assays and viral infections because these cultures are prone to contamination 3 Store at 4 C C Establishing the Sf21 Cell Line 1 Add 5 ml of BacPAK Complete Medium to a 25 cm flask warm to 27 C 2 Remove a vial of cells from liquid nitrogen 3 Thaw rapidly by briefly dipping the vial in a 37 C water bath or by rolling the vial in the palm of your hand Keep at room temperature Note Do not continue to warm the cells after they have thawed Heating cells above 28 C will kill them 4 Immerse or thoroughly swab the vial with 70 ethanol to decontaminate the outside 5 In a laminar flow hood transfer the cell suspension to the pre warmed flask Incubate at 27 C for 1 3 hr to allow cells to attach Do not incubate for more than 12 hr 6 When a significant fraction of the cells have attached gently remove the medium and replace with 5 ml of fresh prewarmed 27 C medium 7 Incubate at 27 C until a nearly confluent monolayer forms 7 days We recommend checking the flasks for confluency every other day D Subculturing Sf21 Monolayers 1 Examine cell monolayers under an inverted microscope to check that the cells are healthy and ready for passaging The monolayer should be 80 90 confluent Notes e Sf21 cells are not susceptible to contact in
19. atant at higher for no plaques dilutions such as 107 amp 105 Low transfection efficiency Use undiluted cotrans fection medium in plaque assay OR use medium har vested from cotransfection after 4 5 days Also try a control cotransfection using pBacPAK8 GUS Control transfection generates virus ex Too much or too little plasmid Check DNA concentration pressing GUS but DNA was used of experimental transfer no virus produced vector in the experimental cotransfaction Experimental transfer vector Clean your plasmid prep on DNA contains impurities that a CHROMA SPIN 400 inhibit transfection column Control transfection does not generate Viral DNA damaged by Handle viral DNA gently do virus expressing shearing not vortex GUS medium does not turn blue with Transfection inhibited by Wash soak and wash cells X Gluc serum or components in the in medium that is protein media and serum free Viral DNA or Bacfectin Fresh batches of Bacfectin damaged by freezing and BacPAK6 viral DNA Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual Vill Construction of a Recombinant Vector continued C Isolating Recombinant Viruses Most viruses in the cotransfection supernatant will be recombinant so you can use it as the primary stock of recombinant virus This stock can be amplified titered Section IX and used to exp
20. em User Manual Appendix A Vector Maps and MCS Seguences continued BamH Sse8387 I Pstl Stu Am pBacPAK8 GUS 7 4 kb signal Xma 1433 bp Smal M13 ori AEM Eagl Not Pacl Figure 7 Map of pBacPAK8 GUS Transfer Vector Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc SeaPlaque is a registered trademark of FMC Corp NucleoBond is a registered trademark of Macherey Nagel GmbH and Co Clontech the Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Clontechis aTakara Bio Company 2008 Clontech Laboratories Inc Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558
21. eserved cell suspension to seed a 50 ml suspension culture Section V E Maintain monolayer stocks of cells by repeating Steps 8 14 You must passage monolayers split 1 8 and grown at 27 C every 3 4 days you must passage monolayers split 1 10 and grown at room temperature once a week Depending upon your needs you may split near confluent monolayers at any ratio between 1 2 and 1 20 As needed seed additional monolayer flasks and suspension cultures to provide cells for experiments For additional information on seeding densities see Table I Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued TABLE I GUIDELINES FOR SEEDING DENSITIES Size of vessel Number of cells Volume of media 25 cm flask 1 0 x 108 5 ml 75 cm flask 3 0 x 106 10 ml 150 cm flask 6 0 x 108 30 ml Spinner shake flasks 2 0 x 105 ml 50 500 ml E Suspension Cultures of Sf21 Cells Suspension cultures using either spinner or shake flasks are easy to maintain and reproducibly give cells of a high viability gt 95 that are good for experimental work Suspension cultures are particularly useful when large numbers of cells are needed Growing cells in spinners requires a low speed magnetic stir platform which can be placed inside a27 C incubator Note that some stir platforms generate too much heat to be used inside a
22. ge 134 kb size of the ACMNPV genome Ayres et al 1994 makes direct manipulation of it difficult so recombinant baculovirus expression vectors are constructed in two steps Figure 1 First a target gene is cloned into a modified polyhedrin locus contained in a relatively small transfer vector lt 10 kb The polyhedrin coding sequence has been deleted and replaced with a multiple cloning site MCS A target gene is inserted into this MCS between the polyhedrin promoter and polyadenylation signals Transfer vectors also contain a plasmid origin of replication and an antibiotic resistance gene for propagation in E coli but they are unable to replicate in insect cells In the second step the transfer vector and a viral expression vector are cotransfected into insect cells Double recombination between viral sequences in the transfer vector and the corresponding sequences in the viral DNA transfers the target gene to the viral genome The BacPAK Baculovirus Expression System uses BacPAK6 a specially engineered virus that facilitates construction and selection of recombinant expression vectors BacPAK6 has an essential gene adjacent to the polyhedrin locus that provides selection for recombinant viruses Kitts amp Possee 1993 Figure 1 Sites for Bsu36 I which does not cut wild type ACMNPV DNA were introduced into the genes flanking the polyhedrin expression locus Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Vers
23. hibition If monolayers become overconfluent the cells will start to float and divide in the media e Healthy cells should be rounded have distinct cell boundaries and should not appear granular Signs of unhealthy cells are a large number of floating cells before confluency sausage shaped cells stopped in mid cell division and cells with rough boundaries Contaminated cultures will become cloudy within 24 48 hr 2 Remove the old medium and any floating cells from the flask If the cells are mainly detached omit this step and go to Step 4 3 Add 5 ml of prewarmed BacPAK Complete Medium Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued 4 10 11 12 13 14 15 16 Gently dislodge the cells using a sterile scraper Note Many commercial scrapers are harsh and using them may result in significant numbers of dead cells Nunc scrapers are acceptable but the best ones are made by attaching a piece of silicon rubber tubing to a bent glass rod Alternatively wash cells using a stream of medium from a pipette Sf21 cells attach less strongly to glass and passaging them is easier if you use glass tissue culture flasks Disperse the cells by gently pipeting up and down 3 4 times Transfer the cell suspension to a 150 cm flask containing 30 ml of prewarmed BacPAK Complete Medi
24. iley Liss Inc NY pp 91 128 Webb A C Bradley M K Phelan S A Wu J Q amp Gehrke L 1991 Use of the polymerase chain reaction for screening and evaluation of recombinant baculovirus clones BioTechniques 11 512 519 Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual Appendix A Vector Maps and MCS Seguences AlwN I BstX I MCS 4950 314 Bam 1 1253 Mlu Sse8387 Pstl Stu l Xho BstB Xbal Bgl ll P Amp pBacPAK8 Polyhedrin FUN sp Kpn 5 5 kb poly At E signal AS cN36 ll 1433 bp Hinam Sel AcMNPV 1764 EcoR Xma Smal Eagl Dra lll Noti 3075 Pac 1325 1250 bp AcMNPV Scal 3990 1401 M13 ori Figure 3 Map of pBacPAK8 Transfer Vector 1151 Bac1 Primer TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA EcoRV 1201 1 polyhedrin promoter AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA 1251 CGGATCCCTG CAGGCCTCGA GTTCGAATCT AGAAGATCTG GTACCGAGCT BamHI Stul BstBl Xbal Bglll Asp718l Ecl136ll Sse83871 Xhol Kpnl Sacl 1301 Pstl stops IT _CGAATTCCCG GGCGGCCGCT TAATTAATTG ATCCGGGTTA TTAGTACATT EcoRI Eagl Pacl Notl Xmal 1351 Smal TATTAAGCGC TAGATTCTGT GCGTTGTTGA TTTACAGACA ATTGTTGTAC GCG ATCTAAGACA CGCAACA SnaBl Pe 1401 Bac2 Primer GTATTTTAAT AATTCATTAA ATTTATAATC Figure 4
25. imum harvest time and infection ratio that will give maximum protein yield Section X Scale up produce target protein in large quantities by infecting larger batches of insect cells Section XI Clontech Laboratories Inc www clontech com Protocol No PT1260 1 8 Version No PR862558 BacPAK Baculovirus Expression System User Manual IV Experimental Outline continued Establish Prepare target vector Section VII S121 cells Insert target gene into transfer vector Section V C e Verify correct construct a Ny Plasmid Preparation Freeze cells Maintain working for long term stocks of Sf21 cells BacPAK6 storage Sections V D amp V E Plasmid DNA Viral DNA Section V F F Practice ae plaque assay Section VI A Cotransfect Sf21 cells with plasmid DNA and BacPAK6 viral DNA Section VIII A U Plague assay of progeny viruses Section VIIIC amp VI A U Pick several putative recombinant virus plagues amp Confirm presence and or expression of target gene Sections IX A amp B U Amplify recombinant virus KTM You may use theBacPA 4 Sections IX C amp D Baculovirus Rapid Titer Kit il Cat No 631406 at steps IX C amp IX E Titer amplified virus stock Section IX E U Characterize gene expression Section X U Scale up protein production Section XI Figure 2 Schematic diagram outlining BacPAK Baculovirus Expression Procedures Protocol No PT1260 1 ww
26. iny upon exposure to light Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual VI Plague Assay Method continued B Calculation of Virus Titer 1 Count the plaques on each dish that has a reasonable number of plaques i e 10 30 per dish from this count calculate the average number of plaques per dish 2 Since 0 1 ml of inoculum was applied to each dish the titer of the virus stock pfu ml is average plaques per dish x 10 x dilution factor 3 Example calculation 25 plaques x 10 x 105 2 5 x 10 pfu ml C Troubleshooting Plaque Assays To get good plaque formation it is important to use cells which are in the exponential phase of growth and are gt 90 viable The density at which the cells are seeded for the plaque assay is also critical Problem Cause Solution Cells are dead Agarose overlay may have Be sure to cool agarose whole plate is been too hot to 37 42 C before use uniformly red Virus inoculum may have been Try higher dilutions too high resulting in complete lysis of the cells Plagues are very Cells may have been seeded Seed dishes with fewer small or invisible too densely cells Plagues are large Cells may have been seeded Seed dishes with more but diffuse too sparsely cells Plagues appear There may have been some Be sure to aspirate all of smeared liguid under the agarose the virus
27. ion No PR862558 BacPAK Baculovirus Expression System User Manual I Introduction continued r ori amp Transfer vector with insert gt ESSENTIAL GENE X X E SENTIAL GENE A Digested BacPAK6 viral DNA Recombination Recombinant baculovirus ESSENTIAL GENE gt Polyhedrin promoter Figure 1 Transfer of a target gene to the baculovirus expression vector by forced recombina tion between a transfer vector and BacPAK6 viral DNA of BacPAK6 Digesting BacPAK6 with Bsu36 I releases two fragments The first carries part of a downstream gene ORF1629 that is essential for viral replication Possee et al 1991 If the second large DNA fragment recircularizes by itself the resulting viral DNA will lack an essential part of the genome and be unable to produce viable viruses However the transfer vector carries the missing ORF1629 seguence and if the large fragment recombines with it the resulting circular DNA will contain all the genes necessary for viral replication This double recombination event restores the essential gene and transfers the target gene from the transfer vector to the viral genome Cotransfections using Bsu36 I digested BacPAK6 viral DNA produce recombinant viruses at frequencies approaching 100 This User Manual contains directions for establishing insect cell cultures as well as for isolating a recombinant baculovirus expression vector using the BacPAK system More exte
28. ith 5 x 10 Sf21 cells in 1 5 2 5 ml of BacPAK Complete Medium Incubate at 27 C for 1 6 hr 2 Remove medium from cells Gently add 100 ul of a plaque pick near the center of the dish As controls plate 100 ul of medium and 100 ul of a 10 dilution of the BacPAK6 parental virus stock 3 Incubate at room temperature for 1 hr 4 Add 2 ml of BacPAK Complete Medium to each dish 5 Incubate at 28 C for 3 4 days until the cells look well infected Infected cells may be grainy sausage shaped or have rough borders 6 Transfer medium to a sterile centrifuge tube Do not discard the dish you will use the cells later 7 Centrifuge medium at 1000 x g for 5 min to remove cells and debris 8 The supernatant is the Passage One virus stock Transfer it to a fresh sterile tube Store at 4 C See Section IX C 1 for further processing of the Passage One virus stock B Evaluation of Recombinant Viruses Many screening methods can confirm that plaques picked from the cotransfection contain recombinant baculovirus The probes available will dictate the method you choose The preferred methods detect synthesis of the target protein e g Western blotting ELISA or a biochemical assay for the expressed protein If an antibody is not available Southern blotting with a nucleic acid probe or PCR with the Bac1 and Bac2 Primers O Reilly etal 1992 Webb et al 1991 Malitschek amp Schartl 1991 Sisk et al 1992 can confirm that the ta
29. l 20 30 ml 25 cm flask 2 0 1 5 x 108 0 4 1 0 ml 3 0 5 0 ml 75 cm flask 6 0 4 0 x 108 0 1 3 0 ml 10 15 ml 150 cm flask 12 0 8 0 x 108 2 0 6 0 ml 20 30 ml When infecting cells for protein production the object is to get all the cells infected synchronously Therefore a high M O I multiplicity of infection is used Initially an M O I of 10 is recommended but you may also want to try M O I s of 5 and 20 Most proteins expressed from the polyhedrin promoter reach their maximum levels somewhere between 24 hr and 60 hr post infection the best time to harvest depends on the nature of the target protein Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual XI Large scale Target Protein Production The preferred method for producing target protein is to infect cells grown in monolayer culture since it is easier to achieve synchronous infections in monolayer cultures than in suspension cultures However if you need more target protein than can practically be produced in a series of 150 mm dishes or 150 cm flasks you may want to use suspension cultures To prepare suspension cultures for large scale protein production seed 100 500 ml suspension cultures with 2 x10 Sf21 cells ml and infect them by adding the requisite volume of virus stock when they reach 1 x 106 cells ml To achieve maximal protein expression use BacPAK Complete Medium o
30. ment of baculovirus expression vectors Bio Technology 6 47 55 Malitschek B amp Schartl M 1991 Rapid identification of recombinant baculoviruses using PCR BioTechniques 11 177 178 Miller L K 1988 Baculoviruses as gene expression vectors Ann Rev Microbiol 42 177 199 O Reilly D R Miller L K amp Luckow V A 1992 Baculovirus Expression Vectors A Laboratory Manual W H Freeman amp Co NY Possee R D 1986 Cell surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector Virus Res 5 43 59 Possee R D Sun T P Howard S C Ayres M D Hill Perkins M amp Gearing K L 1991 Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9 4 kbp EcoR l I and R polyhedrin gene region Virol 185 229 241 Richardson C D 1995 Baculovirus Expression Protocols Methods in Molecular Biology Volume 39 Humana Press NJ Sisk W P Bradley J D Seivert L L Vargas R A amp Horlick R A 1992 An improved method for rapid screening of baculovirus recombinant plaques by PCR amplification BioTechniques 13 2 186 Vaughn J L Goodwin R H Tompkins G J amp McCawley P 1977 The establishment of two cell lines from the insect Spodoptera frugiperda Lepidoptera Noctuidae In Vitro 13 213 217 Vlak J M amp Keus R J A 1990 Baculovirus expression vector system for production of viral vaccines in Viral Vaccines W
31. n incubator Flat bottomed pyrex flasks 100 1000 ml containing a magnetic stir bar and covered with a foil cap can be used as spinner flasks Alternatively suspension cultures can be grown in shake flasks using an orbital shaker normally used for bacterial cultures 1 Add an appropriate volume of prewarmed BacPAK Complete Medium to a sterile spinner or shake flask Inoculate with cells to give a starting density of 4 x 10 cells ml Note Insect cells have a high oxygen demand therefore suspension cultures must have a high surface area to volume ratio or cell growth will be inhibited The culture volume should be no more than two fifths of the total volume of the flask e g 40 ml of medium in a 100 ml flask or 100 ml in a 250 ml flask 2 Incubate cells at 27 C and stir or shake at 50 100 rpm use the minimum speed that will keep the cells in suspension 3 Monitorthecell density daily untiltheculturereaches2 3x106cells ml 4 days Add 0 3 ml of cell suspension to 0 3 ml of 0 08 w v trypan blue in PBS Count the cells using a hemocytometer viable cells exclude trypan blue whereas dead cells take up the blue stain 4 Use the cells to seed a fresh spinner shake flask at a density of 1 2 x 10 cells ml Alternatively remove the excess cells and add fresh media to bring the density down to 1 2 x 10 cells ml 5 Return to stirrer shaker at 27 C and monitor cell density daily as above Protocol No PT1260 1 www clon
32. neration of recombinant viruses expressing GUS The negative control dish should not change color 5 days after addition of the Bacfectin DNA mixture to the cells transfer the medium which contains viruses produced by the transfected cells to a sterile container and store at 4 C Optional To obtain more virus add a fresh aliquot 1 5 ml of BacPAK Complete Medium to each dish Incubate at 27 C for another 2 days and harvest the medium as above Clontech Laboratories Inc www clontech com Protocol No PT1260 1 Version No PR862558 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Vector continued B Troubleshooting Guide Problem Cause Solution Medium on trans Microbial contamination due Use freshly autoclaved H 0 fected cells turns to contaminated materials or pipette tips etc Sterilize cloudy poor sterile technique plasmid DNA by ethanol precipitation To rescue pass medium through a 0 2 uM sterile filter and plaque assay the filtrate or you may add gentamycin pen strep to the medium Few or no viral Poor plaque assay Plaques Practice assay Make sure plaques recovered on control plates will also be you use healthy cells from cotransfection small or invisible Dilutions were inappropriate Carefully compare with for titer of virus High virus plates with medium only titer will produce confluent Plate the cotransfection plaques that may be mistaken supern
33. nsive protocols for using baculovirus expression systems are in the baculovirus laboratory manuals O Reilly et al 1992 King amp Possee 1992 Richardson 1995 Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 5 BacPAK Baculovirus Expression System User Manual ll List of Components Store the following item at 1809C liguid nitrogen immediately upon receipt 2 x 109 IPLB Sf21 Insect Host Cells in TNM FH 10 FBS 10 DMSO Store the following items at 4 C do not freeze The following components are sufficient for five transfections e 25 ul BacPAK6 Viral DNA Bsu36 digest e 25 ul Bacfectin For long term storage of 6 months or longer store the following reagents at 70 C For storage less than 6 months store at 4 C protected from light e 2 ml BacPAK6 Virus Stock For long term storage store the following reagents at 20 C For storage less than 6 months store at 4 C e 15 ug pBacPAK8Transfer Vector 500 ng ul e 15 ug pBacPAK9Transfer Vector 500 ng ul e 2 5 ug pBacPAK8 GUS Vector 100 ng ul e 20 ul Bact Primer 20 uM e 20 ul Bac2 Primer 20 uM Note The following kit component is also available separately e BacPAK6 DNA Bsu36 digest Cat No 631401 Clontech Laboratories Inc www clontech com Protocol No PT1260 1 6 Version No PR862558 BacPAK Baculovirus Expression System User Manual lll Additional Materials Reguired The following mate
34. r other high quality medium and fetal bovine serum and log phase Sf21 cells that are at least 98 viable Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual XII References Ayres M D Howard S C Kuzio J Lopez Ferber M amp Possee R D 1994 The complete DNA sequence of Autographa californica nuclear polyhedrosis virus Virology 202 586 605 Bishop D H L amp Possee R D 1990 Baculovirus expression vectors Advances GeneTechnol 1 55 72 Grace T D C 1962 Establishment of four strains of cells from insect tissues grown in vitro Nature 195 788 789 Hink W F 1970 Established insect cell line from the cabbage looper Trichoplusia ni Nature 226 466 467 King L A amp Possee R D 1992 The Baculovirus Expression System A Laboratory Guide Chapman amp Hall NY Kitts P A 1996 Construction of baculovirus recombinants Cytotechnology 20 111 123 Kitts P A amp Possee R D 1993 A method for producing recombinant baculovirus expression vectors at high frequency BioTechnigues 14 5 810 817 Luckow V A 1991 Cloning and expression of heterologous genes in insect cells with baculovirus vectors in Recombinant DNATechnology amp Applications eds Prokop A Bajpai R K amp Ho C S McGraw Hill Inc NY pp 97 152 Luckow V A amp Summers M D 1988 Trends in the develop
35. r vectors having the insert in the correct orientation by PCR amplification using the Bac1 and Bac2 Primers or by performing restriction digests of mini prep DNA Optional Confirm the integrity of the junctions by sequencing with the Bac1 and Bac2 Primers Prepare plasmid DNA by CsCl isopycnic density gradient centrifugation or by alkaline lysis miniprep followed by purification with a CHROMA SPIN TE 400 Column You may also use a NucleoBond Plasmid Purification Kit Cat Nos 740571 740571 100 Clontech Laboratories Inc www clontech com Protocol No PT1260 1 18 Version No PR862558 BacPAK Baculovirus Expression System User Manual VIII Construction of a Recombinant Viral Expression Vector A Generating a Recombinant Virus Vector DNA e g pBacPAK8 or pBacPAK9 containing the target gene is transfected into Spodoptera frugiperda cells along with Bsu361 digested BacPAK6 Viral DNA In vivo homologous recombination between the plasmid and viral DNA rescues the viral DNA and transfers the target gene to the viral genome Kitts amp Possee 1993 Kitts 1996 You may use pBacPAK8 GUS asa positive control forthe cotransfection step This transfer vector has the E coli B glucuronidase GUS gene cloned downstream of its polyhedrin promoter Recombination of pBacPAK8 GUS with BacPAK6 DNA digest generates recombinant viruses that express B glucuronidase Expression of GUS can be detected by generation of a blue
36. refer to Figure 2 on the following page Obtain insect cell media and establish Sf21 cell line This step will take 3 4 weeks Section V B C Maintain working stocks of Sf21 cells Sections V D E When the stock of cells has been passaged twice freeze aliquots for long term storage in liquid nitrogen Aliquots of frozen cells provide a back up in case the working stock dies or becomes contaminated Frozen cells are also a source of fresh cells for replacing working stocks as they become old Section V F Practice assaying viral plaques using the BacPAK6 virus stock provided in the kit Section VI A Isolating pure recombinant virus requires good viral plaques Therefore developing a good plaque assay technique before working with recombinant viruses is advisable Insert target gene into transfer vector Section VII and prepare plasmid DNA Produce arecombinant virus by cotransfecting Sf21 cells with BacPAK6 viral DNA and the transfer vector target gene clone Section VIII A Perform plaque assays on the cotransfection supernatant to obtain individual viral plaques Section VIII C Test the putative recombinant viruses to confirm that they have incorporated the target gene and or express the target protein Section IX A B Amplify recombinant viruses to obtain working stocks Section IX C D Titer amplified virus stock Section IX E Perform small scale infections to characterize gene expression and to determine the opt
37. ress protein Sections X amp XI However it will contain a mixture of viruses and its composition may change with repeated passage resulting in altered expression This short cut can be used to guickly produce a few batches of protein If many batches of protein are to be produced a clonal stock of virus should be produced to ensure consistency You obtain a pure clone of a recombinant virus by diluting the cotransfection supernatant containing progeny viruses and doing a plague assay to produce individual plagues Optional The positive control cotransfection can be assayed to determine the yield and proportion of recombinant viruses expressing GUS In this case X gluc is added to the overlay in a plague assay of the control cotransfection supernatant Steps 7 amp 11 1 2 Seed fourteen 35 mm dishes with 1 x 108 Sf21 cells in 1 5 ml of medium and incubate at 27 C for 1 4 hr Make serial dilutions of the cotransfection supernatant 100 ul into 900 ul of BacPAK Complete Medium to give final dilutions of 101 10 2 and 103 Toprovide a positive control dilutethe BacPAK6 virus stockprovided so that 100 ul will produce 10 30 plaques on a dish this will be a dilution between 10 and 10 Inspect the dishes from Step 1 above to ensure that the cells have attached to form an even monolayer of about 70 80 confluency Aspirate the medium from the cells using a sterile pasteur pipette or pipette tip Infect
38. rget gene is in the viral genome Detailed screening protocols will not be presented here as they follow standard methods 1 After removing medium from the virus infected cell cultures Step IV A 6 add 1 ml of PBS to each dish and scrape cells into the buffer 2 Pellet the cells in a microcentrifuge at 1 000 rpm for 1 min 3 Remove supernatant and gently resuspend cells in 0 5 ml of PBS 4 Repellet and discard supernatant Analyze cell proteins or DNA as follows Protocol No PT1260 1 www clontech com Clontech Laboratories Inc Version No PR862558 25 BacPAK Baculovirus Expression System User Manual IX Virus Propagation and Evaluation continued 5 Analysis of virus infected cell proteins a b Resuspend the cell pellet in 50 100 ul of PBS Add an appropriate volume of SDS PAGE dissociation mix and boil for 5 min You may run an aliquot of the denatured proteins on a standard polyacrylamide gel and analyze by Western blotting 6 Analysis of virus infected cell DNA a b Resuspend the cell pellet in 250 ul of TE buffer Add 250 ul of lysis buffer 12 5 ul of 10 mg ml proteinase K and 2 ul of 10 mg ml RNase Incubate at 37 C for 30 min Add 500 ul of phenol chloroform 50 50 Mix by inversion for 5 min Spin in a microcentrifuge for 2 min to separate the phases Move the aqueous layer to a fresh tube repeat the extraction twice Transfer the aqueous layer to a fresh tube add 50 ul of 3
39. rials are reguired but not supplied BacPAK Complete Medium Cat No 631403 You may also use TNM FH insect cell medium Grace s medium supplemented with yeastolate and lactalbumin hydrolysate with fetal bovine serum cell culture grade ask vendor for a lot tested with insect cells and antibiotics BacPAK Grace s Basic Medium Cat No 631404 CHROMA SPIN 4 TE 400 Columns Cat No 636076 Dimethylsulfoxide DMSO cell culture grade Neutral red stain 0 33 Trypan blue dye 0 4 X GAL 25 mg ml 5 bromo 4 chloro 3 indolyl p D galactopyranoside in dimethylformamide DMF Store away from light at 20 C X GLUC 25 mg ml 5 bromo 4 chloro 3 indolyl B D glucuronic acid Cat No 631721 in DMSO Store away from light at 209C RNase A 10 mg ml Store at 20 C Proteinase K 10 mg ml made fresh Cat No 635919 Store at 20 C SeaPlaque Agarose Cambrex Cat No 50101 Sterile HO 3 M NaOAc pH 5 2 Lysis buffer 50mM Tris HCI pH 8 0 10mM EDTA 5 B mercaptoethanol 0 4 Sodium dodecylsulfate Phosphate buffered saline PBS 140mM _ NaCl 27mM KCI 8mM Na HPO 1 5mM KH PO pH 7 3 TE buffer 10mM Tris HCI pH 8 0 1mM EDTA Phenol chloroform 50 50 equilibrated with 100 mM Tris HCI pH 8 0 Ethanol 100 and 70 Protocol No PT1260 1 www clontech com Clontech Laboratories Inc 7 Version No PR862558 BacPAK Baculovirus Expression System User Manual IV Experimental Outline Please
40. t insect cell processed proteins have comparable biological activities and immunological reactivities to proteins expressed in mammalian cells Protein yields from baculovirus systems are higher and costs are significantly lower than in mammalian expression systems The baculovirus expression system can express genes from bacteria viruses plants and mammals at levels from 1 500 mg liter most proteins are expressed in the 10 100 mg liter range although making predictions is difficult The baculovirus most commonly used to express foreign proteins is Autographa californica nuclear polyhedrosis virus AcMNPV Luckow 1991 Vlak amp Keus 1990 Bishop amp Possee 1990 Miller 1988 Luckow amp Summers 1988 O Reilly et al 1992 ACMNPV can be propagated in certain insect cell lines the virus enters the cells and replication begins approximately 6 hours post infection h p i At approximately 20 48 h p i transcription of nearly all genes ceases The viral polyhedrin and p10 genes however are transcribed at high rates The polyhedrin protein is essential for propagation of the virus in its natural habitat however in cell culture polyhedrin is not needed and its coding sequence can be replaced with a sequence for a target protein Hence the powerful polyhedrin promoter can drive high level transcription of the insert resulting in expression of a recombinant protein that can account for over 30 of total cellular protein The lar
41. tech com Clontech Laboratories Inc Version No PR862558 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines continued Notes e Maintain one or more suspension cultures to provide cells for experimental work Cells should only be used for virus infections and plaque assays if they are in the exponential phase of growth usually 0 7 1 x 10 cells ml Periodically every 4 6 weeks replace the working suspension cultures with fresh ones started from monolayer cells e Take good care of your cells the quality of virus plaques and the level of protein production are very dependent on the health of the host cells F Storing Insect Cells in Liquid Nitrogen Freezing aliquots of cells in liquid nitrogen provides a source of fresh cells to replace the working stocks when they become old 1 Monitor the cells in a 150 cm flask to ensure that they are healthy and growing exponentially When the monolayer reaches about 80 confluency remove the old medium add 5 ml of prewarmed BacPAK Complete Medium gently scrape the cells and disperse the cells by gently pipeting up and down Count the cells and ensure that they are at least 90 viable Adjustcell density to 4x 108cells ml with BacPAK Complete Medium Chill the cells to 4 C Prepare an equal volume of BacPAK Complete Medium containing DMSO at 20 v v Chill to 4 C Label cryogenic vials and put them on ice Add the BacPAK
42. um Incubate at 27 C until the cells are barely confluent 3 5 days Examine monolayers to check that the cells are healthy and ready for passaging The monolayer should be 80 90 confluent Remove the old medium and any floating cells If the cells are mainly detached omit this step Add 10 ml of prewarmed BacPAK Complete Medium Gently dislodge the cells using a sterile scraper Disperse the cells by gently pipeting up and down 3 4 times Add 0 3 ml of cell suspension to 0 3 ml of 0 08 w v trypan blue in PBS Count cells with a hemocytometer dead cells take up the blue stain Determine the concentration and proportion of viable cells Note After careful harvesting healthy monolayer cells from plastic flasks should have viabilities of 80 90 monolayers harvested from glass flasks should have viabilities of gt 90 and suspension cultures should have viabilities of gt 95 Remove all but 2 ml of the 10 ml cell suspension and store it in a sterile container Add 30 ml of prewarmed BacPAK Complete Medium to the remaining 2 ml of culture Swirlto mix and incubate at 27 C For information on incubation times see note below Add 2 ml of the reserved cell suspension to a second 150 cm 2 flask containing 30 ml of prewarmed BacPAK Complete Medium Swirl to mix and incubate at 27 C The cells from this flask will be frozen Section V F For information on incubation times see note below Use a portion of the remaining r
43. w clontech com Clontech Laboratories Inc Version No PR862558 9 BacPAK Baculovirus Expression System User Manual V Insect Cell Culture Guidelines A General Considerations The IPLB Sf21 cell line abbreviated Sf21 originally derived from the fall army worm Spodoptera frugiperda Vaughn et al 1977 is used to propagate AcCMNPV based expression vectors These cells grow reasonably well from room temperature 22 C to 30 C and do not require CO At their optimum growth temperature 27 C their doubling time is 20 24 hr Although you can culture Sf21 cells on the bench maintaining them in an incubator at 27 C is preferable for consistent virus infections You may culture them as monolayers or in suspension cells can be freely transferred between the two culture types To maintain consistency do not passage cells indefinitely After 20 30 passages replace a culture with fresh cells from liquid nitrogen To prevent contamination work with media and uninfected cells in a vertical laminar flow hood using sterile technique Keep this hood free of virus to avoid accidental infection of the stock cultures ideally use another hood for all virus work Virus infections can be performed on the bench following good microbiological practice unless the recombinant virus carries a potentially harmful or infectious gene Although baculoviruses have a restricted host range treat recombinant baculoviruses as potential biohazards All
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