5.4 Cleaving Fusion Proteins using Site-specific Proteases . ...... ting sites have been added, in ORF 603, 1629 and in lacZ, which do not alter the amino acid ...
6th Edition, Edition, May May 1999 1999 6th
Expression Vector System
Baculovirus
Instruction Manual
Baculovirus Expression Vector System Manual 6th Edition
May 1999
Instruction Manual
General Methods 6xHis and GST Purification Direct Cloning
For information or to place an order, please call: 1-800-848-MABS (6227) For Technical Assistance call: 1-800-TALK-TEC (825-5832) 10975 Torreyana Road • San Diego, CA 92121 • USA Tel: (619)812-8800 • Fax: (619) 812-8888 URL: http://www.pharmingen.com
Table of Contents Baculovirus Memorandum of Agreement . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
Opening Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vii
1. The Baculovirus Expression Vector System . . . . . . . . . . . . . . . . . . . . . . . .
1
2. Advantages of using the Baculovirus Expression Vector System . . . . . . .
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3. AcNPV Baculovirus DNAs . . . . . . . . . . . . . . . . AcNPV C6 Wild-type Baculovirus DNA . . . BaculoGold™ Linearized Baculovirus DNA Linearized AcRP23.lacZ Baculovirus DNA . Linearized AcUW1.lacZ Baculovirus DNA .
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4. General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Selecting an Appropriate Baculovirus Transfer Vector . . . . . 4.2 Optimizing Gene Expression . . . . . . . . . . . . . . . . . . . . . . . 4.3 Cloning your Gene into a Baculovirus Transfer Vector . . . . Preparing Vector and Insert . . . . . . . . . . . . . . . . . . . . . . . . Ligating Vector and Insert . . . . . . . . . . . . . . . . . . . . . . . . . Propagating Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purifying Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Insect Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Handling Techniques . . . . . . . . . . . . . . . . . . . . . . . Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Freezing and Thawing Insect Cells . . . . . . . . . . . . . . . . . . . 4.5 Producing and Maintaining AcNPV-derived Baculoviruses . Generating Recombinant Baculoviruses by Co-Transfection End-point Dilution Assay . . . . . . . . . . . . . . . . . . . . . . . . . . Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plaque Pickup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amplifying Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Storing Virus Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolating AcNPV Particles . . . . . . . . . . . . . . . . . . . . . . . . . . Isolating AcNPV DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Expressing Recombinant Proteins . . . . . . . . . . . . . . . . . . . . Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Purifying Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . Non-secreted Recombinant Proteins . . . . . . . . . . . . . . . . . . Cell Lysate Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secreted Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . .
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iii
5. Purification Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 6xHis Expression and Purification Kit . . . . . . . . . . . . . . Batch Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 GST Expression and Purification Kit . . . . . . . . . . . . . . . Batch Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . Dialyzing GST-Fusion Protein . . . . . . . . . . . . . . . . . . . . 5.3 Checking Purity and Recovery of Recombinant Protein 5.4 Cleaving Fusion Proteins using Site-specific Proteases . . Thrombin Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . Factor Xa Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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39 39 41 42 43 44 45 45 46 46 46
............ 5.5 Generating 32P-Labeled GST or 6xHis Fusion Proteins . . . . . . . . . . . . . .
47 47
6. Generating Recombinant Baculovirus by Direct Cloning . . . . . . . . . . . .
49
7. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Cloning Inserts into Baculovirus Transfer Vectors 7.2 Insect Cell Culture . . . . . . . . . . . . . . . . . . . . . . . 7.3 Co-transfection . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Virus Amplification . . . . . . . . . . . . . . . . . . . . . . 7.6 Recombinant Protein Production . . . . . . . . . . . . 7.7 6xHis Expression and Purification System . . . . . 7.8 GST Expression and Purification System . . . . . . . 7.9 Thrombin Cleavage . . . . . . . . . . . . . . . . . . . . . . 8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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53 53 53 54 55 56 56 57 58 59 61
Appendix A: BaculoGold™ Starter Package and Transfection Kit . . . . . . . . . .
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Appendix B: 6xHis Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Appendix C: GST Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Appendix D: vEHuni and vECuni Baculovirus Reagent Sets . . . . . . . . . . . . . .
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Appendix E: Baculovirus Transfer Vectors I. Polyhedrin Locus-based Vectors . . . . . . Fusion Vectors . . . . . . . . . . . . . . . BioColors™ Baculovirus Vectors . . Multiple Promoter Transfer Vectors II. p10 Locus-based Vectors . . . . . . . . . . . . Multiple Promoter Transfer Vectors Index
iv
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. 77 . 77 . 82 . 92 . 96 . 100 . 101
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Figures 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
The Baculovirus life cycle in vivo and in vitro . . . . . . . . . . . . . . . . . . . . Design of AcNPV BaculoGold™ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . Design of AcRP23.lacZ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Design of AcUW1.lacZ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental scheme using BEVS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monolayer and suspension Sf cultures . . . . . . . . . . . . . . . . . . . . . . . . . Comparison of uninfected and infected Sf9 cell monolayers . . . . . . . . 12-well End-point Dilution Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Western blot analysis of Retinoblastoma protein (Rb) in plaques . . . . . Examples of recombinant protein expression levels in Baculovirus-infected Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of native and Baculovirus-expressed Retinoblastoma protein (Rb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional activity of Baculovirus-expressed recombinant protein . . . . Expression, purification and cleavage of fusion proteins . . . . . . . . . . . Strategy for directly cloning EcoRI fragments into the AcMNPV genome Baculovirus vectors for direct cloning . . . . . . . . . . . . . . . . . . . . . . . . . BioColors™ in Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Separation of Baculovirus-expression GFP and BFP using fluorescence-activated cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Tables 1. 2. 3. 4.
Comparison of BEVS and bacterial expression systems . . . . . . . . . . . . . . Analysis of recombination frequencies by plaque assays . . . . . . . . . . . . . . Vector selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended cell numbers and approximate densities for various assays
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v
BACULOVIRUS MEMORANDUM OF AGREEMENT NON-EXCLUSIVE RIGHTS TO USE BACULOVIRUS EXPRESSION VECTOR SYSTEM TECHNOLOGY FOR RESEARCH PURPOSES I. BACKGROUND The Texas Agricultural Experiment Station (TAES) claims rights to technology developed by Dr. Max D. Summers of the Department of Entomology relating to a recombinant Baculovirus expression vector system (BEVS) and the use of such vectors in insect cell culture media for expression of cloned genetic material. TAES is making the system and its components available for noncommercial research purposes. This Baculovirus expression vector system and related subject matter are claimed in two United States Patents, Numbers 4,745,051 and 4,879,236. Commercial rights to BEVS or products thereof are subject to a non-exclusive license, terms of which will be made available upon written request. Information and materials received from TAES relating to BEVS must be taken with the understanding that it is subject to a restrictive license for research purposes only. II. TERMS AND CONDITIONS OF AGREEMENT (1)
All information and material received under this Agreement shall be used for research purposes only.
(2)
Access and distribution of the vectors and information must be limited to Recipient and to those personnel who report to Recipient, hereinafter referred to as "Recipient."
(3)
Recipient agrees to supply TAES preprints of any publications resulting from the use of the BEVS material promptly upon receipt of notice of acceptance from the publishing journal. Preprints should be sent to the attention of the Coordinator of Research Development for Industrial Relations, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843-2162.
(4)
Recipient and those who report to Recipients are aware of the proprietary interest involved herein and commit to honoring the terms and conditions of this Agreement.
(5)
Recipient accepts the biological material with the knowledge that it is experimental biological material and that is provided by TAES without warranty of any sort, expressed or implied. Recipient agrees to comply with all applicable governmental regulations for the handling thereof. Recipients shall hold TAES harmless for any damages which may be alleged to result in connection with the use and possession of the requested materials as provided in this Agreement, subject to any relevant state or federal government limitations.
(6)
This Agreement and Recipient’s right to use biological material become effective upon breaking the seal of the package containing biological material and automatically terminates if Recipient fails to comply with any provisions of this Agreement.
(7)
TAES retains ownership and all rights to biological material not expressly granted and nothing in this Agreement constitutes a waiver of TAES' rights under U.S. Federal, State, or Patent law.
NOTE: THESE RESTRICTIONS DO NOT APPLY TO INFORMATION OR TECHNOLOGY WHICH RECIPIENT CAN SHOW ARE IN THE PUBLIC DOMAIN OR FOR WHICH HE/SHE HAD PREVIOUSLY RECEIVED OR DEVELOPED IN GOOD FAITH THROUGH CHANNELS INDEPENDENT OF THE TEXAS AGRICULTURAL EXPERIMENT STATION.
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Opening Remarks All reagents and materials listed in this manual are for research use only. Safety Requirements These research products have not been approved for human or animal diagnostic or therapeutic use. We suggest that all purchasers follow the NIH guidelines that have been developed for recombinant DNA experiments. All PharMingen products should be handled only by qualified persons trained in laboratory safety procedures. The absence of a product warning is not to be construed as an indication that the product is safe. All possible hazards of many biological products may not be known at this time. Always use good laboratory procedures when handling any of these products. Warranty Information presented in this manual is accurate to the best of our knowledge. It is not, however, guaranteed as such. It is the user’s responsibility to investigate and verify the suitability of the supplied materials and procedures for a particular purpose. PharMingen expressly disclaims all warranties of merchantability and fitness for a particular purpose with respect to the use or suitability of the reagents and materials. PharMingen shall in no event be responsible for damages of any nature, directly or indirectly resulting from the use of the products of these kits. Disclaimer This manual is a practical guide for researchers to become familiar with the Baculovirus expression technology as a tool to overexpress foreign genes. It is not intended as a replacement to a textbook about Baculoviruses but rather to serve as an introduction to Baculovirus nomenclature and cite key references to guide the interested reader to additional literature. The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. PharMingen will not be held responsible for patent infringement or other violations that may occur with the use of our products. For commercial use of the 6xHis/Ni-NTA system, licenses may be granted by Hoffmann-La Roche Ltd. (Basel, Switzerland). Please contact QIAGEN Inc., 9600 De Soto Avenue, Chatsworth, CA 91311 for further information. All Baculovirus and related products sold by PharMingen are for research use only. The Polymerase Chain Reaction (PCR) is a process patented by Hoffmann-La Roche, Inc. Triton is a trademark of Union Carbide Chemicals and Plastics Co. Technical Assistance and Ordering Information At your request, we will furnish technical assistance and information about our products. Call 800-TALK-TEC (825-5832) to talk to a Technical Service Specialist. Our specialists have the education and experience necessary to answer your technical questions regarding the reagents and materials listed in this manual. All technical assistance is provided gratis and you assume sole responsibility for results you obtain by relying on that assistance. We make no warranties of any kind with respect to technical assistance or information we provide. Call 800-848-MABS (848-6227) to place an order.
vii
Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus Amp Ampicillin β-gal β-galactosidase BEVS Baculovirus expression vector system BFP Blue fluorescent protein BSA Bovine serum albumin BV Baculovirus CIAP Calf intestinal alkaline phosphatase CsCl Cesium chloride DTE Dithioerythritol DTT Dithiothreitol ECV Extracellular virus EDTA Ethylenediamine tetraacetic acid EtBr Ethidium bromide FACS Fluorescent activated cell sorting FBS Fetal bovine serum GFP Green fluorescent protein GST Glutathione S-transferase h Hour kb Kilobases kD Kilodalton LB Luria-bertani (broth) MCS Multiple cloning site min Minute MOI Multiplicity of infection (plaque-forming units/cell number) NaPi Sodium phosphate NaPPi Sodium pyrophosphate ORF Open reading frame OV Occluded virus particles PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline pfu Plaque-forming unit(s) = virus Pi Inorganic phosphate pi Post infection PMSF Phenylmethylsulfonyl fluoride Rb Retinoblastoma protein RT Room temperature Sf Spodoptera frugiperda Sj Schistosoma japonicum SDS Sodium dodecyl sulfate TBE Tris borate/EDTA TE Tris/EDTA U Unit v/v Volume: volume ratio wt Wildtype X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside YP Yellow protein yr Year
viii
The 6xHis vectors were developed by PharMingen and produced in collaboration with QIAGEN
1
The Baculovirus Expression Vector System
The Baculovirus Expression Vector System (BEVS) is one of the most powerful and versatile eukaryotic expression systems available.1,2 The BEVS is a helper-independent viral system which has been used to express heterologous genes from many different sources, including fungi, plants, bacteria and viruses, in insect cells. The Baculovirus DNA used in PharMingen’s BEVS is the Autographa californica nuclear polyhedrosis virus (AcNPV). In this system several Baculovirus genes nonessential in the tissue culture life cycle (polyhedrin, p10, basic) may be replaced by heterologous genes. Since the Baculovirus genome is generally too large to easily insert foreign genes, heterologous genes are cloned into transfer vectors.† Co-transfection of the transfer vector and AcNPV DNA into Spodoptera frugiperda (Sf) cells allows recombination between homologous sites, transferring the heterologous gene from the vector to the AcNPV DNA. AcNPV infection of Sf cells results in the shut-off of host gene expression allowing for a high rate of recombinant mRNA and protein production. Recombinant proteins can be produced at levels ranging between 0.1% and 50% of the total insect cell protein. Factors influencing foreign gene expression are discussed, although it is difficult to precisely predict how efficiently different genes will be expressed. Baculoviruses (family Baculoviridae) belong to a diverse group of large doublestranded DNA viruses that infect many different species of insects as their natural hosts.3 Baculovirus strains are highly species-specific and are not known to propagate in any non-invertebrate host. The Baculovirus genome is replicated and transcribed in the nuclei of infected host cells where the large Baculovirus DNA (between 80 and 200 kb) is packaged into rod-shaped nucleocapsids.4 Since the size of these nucleocapsids is flexible, recombinant Baculovirus particles can accommodate large amounts of foreign DNA. AcNPV is the most extensively studied Baculovirus strain. Its entire genome has been mapped and fully sequenced.5-7 Infectious AcNPV particles enter susceptible insect cells by facilitated endocytosis or fusion, and viral DNA is uncoated in the nucleus (Fig. 1). DNA replication starts about 6 h post-infection (pi). In both in vivo and in vitro conditions, the Baculovirus infection cycle can be divided into two different phases, early and late. During the early phase, the infected insect cell releases extracellular virus particles (ECV) by budding off from the cell membrane of infected cells. During the late phase of the infection cycle, occluded virus particles (OV) are assembled inside the nucleus. The OV are embedded in a homogenous matrix made predominantly of a single protein, the polyhedrin protein.8, 9 OV are released when the infected cells lyse during the last phase of the infection cycle. Whereas the first ECV are detectable 10 h pi, the first viral occlusion bodies of wild-type AcNPV virus develop 3 days pi but continue to accumulate and reach a maximum between 5-6 days pi. Occlusion bodies are visible under light microscopy where they appear as dark polygonal-shaped bodies filling up the nucleus of infected cells. Not all known Baculoviruses form occlusion bodies; AcNPV is representative of the group of occlusion body-positive Baculoviruses. The polyhedrin protein, the major component of occlusion bodies, has a molecular weight of 29 kD.1 During late phases of infection, the polyhedrin protein accumulates to very high levels. Up to 1 mg of polyhedrin protein †
vEHuni and vECuni Baculovirus DNAs allow for direct cloning of heterologous genes into the BV genome (Chapter 6).
1
may be synthesized per 1–2 × 106 infected cells accounting for 30-50% of the total insect protein. Although the polyhedrin protein seems to be one of the most abundant proteins in infected insect cells, it is not essential for the Baculovirus life cycle in tissue culture. However, in vivo viral occlusion bodies are an important part of the Baculovirus life cycle, essential for its dissemination into the environment (Fig. 1). Deletional or insertional inactivation of the polyhedrin gene in AcNPV results in the production of occlusion body-negative viruses, a phenomenon which simplifies the identification of recombinant viruses. The plaques formed by occlusion body-negative viruses are distinctly different from those of occlusion body-positive wild-type viruses. Newer modified AcNPV allow either color selection to identify recombinants or permit positive survival selection for recombinants (BaculoGold™ Cat. No. 21001K), rendering the occlusion body-based visual screening method obsolete. A variety of Baculovirus Transfer Vectors have been constructed for use with AcNPV DNA (Appendix E). Each vector contains: 1) an E. coli origin of replication, 2) an ampicillin resistance marker, 3) a promoter from the polyhedrin, p10 or basic protein AcNPV gene,10 4) a cloning region downstream from promoter in which to insert foreign genes and 5) a large tract of AcNPV sequence flanking the cloning region to facilitate homologous recombination. Purified recombinant vectors containing the gene of interest may be co-transfected with AcNPV Baculovirus DNA into insect cells. After several days, recombinant viruses, which arise by homologous recombination between the transfer vector and AcNPV DNA, are selected.
A
B
Secondary Infection of Insect Cells
Budding Virus
Transfer Vecto r You
r Ge ne
Co-transfection
B acu
loGold® DNA
GST/ 6xHIS Tag
Endocytosis
Replication Homologous Recombination
Uncoating
B ac
ul o
Go
ld
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DN A
Y
ou
rG
en e
Viral Occlusion
Fusion (Midgut Cells)
Recombinant Protein
Secondary Infection of Insect Cells
Budding Recombinant Virus
Ingestion Soluble in Gut
Primary Infection of Host Insect
Figure 1. The Baculovirus life cycle in vivo and in vitro. A) In vivo. Two distinct viral populations are formed in infected insect cells, occluded and budded virions. Occluded virions are protected from desiccation in the environment, allowing primary infection in susceptible larva. Once ingested, the occlusion body is solubilized in the gut, releasing virions which fuse with midgut cells. The virion nucleocapsid migrates through the cytoplasm to the nucleus. The core is uncoated from the capsid structure in the nucleus. Here replication takes place. Secondary infection is mediated by the budded form of the virus entering adjacent cells via adsorptive endocytosis. B) In vitro. The Baculovirus genome is too large to directly insert foreign genes easily. Hence, the foreign gene is cloned into a transfer vector that contains flanking sequences which are homologous (5’ and 3’ to your insert) to the Baculovirus genome. BaculoGold™ DNA and the transfer vector containing your cloned gene are co-transfected into Sf9 insect cells. Recombination takes place within the insect cells between the homologous regions in the transfer vector and the BaculoGold™ DNA. Recombinant virus produces recombinant protein and also infects additional insect cells thereby resulting in additional recombinant virus.
2
2
Advantages of using the Baculovirus Expression Vector System
Choosing the right system for foreign gene expression can be particularly important in obtaining biologically active recombinant protein. Several unique features of the BEVS have made it the system of choice for many applications (Table 1). This chapter highlights the advantages of using BEVS to express recombinant proteins. Often, recombinant proteins expressed in bacterial systems are insoluble, aggregated and incorrectly folded.11 In contrast, proteins expressed in BEVS are, in most cases, soluble and functionally active. Features Simple to use Protein size Multiple gene expression Signal peptide cleavage Intron splicing Nuclear transport Functional protein Phosphorylation Glycosylation Acylation
BEVS
Bacterial
✓
✓
unlimited
5,000
1,096
B. AcRP23.lacZ R 1,500 Baculovirus DNA +1392-XylE NR >5,000
>5,000 >5,000 200
52
6
398
87
11
R >5,000 C. BaculoGold™ Baculovirus DNA +1392-XylE NR 3
885
105
9
0
0
0
NA ~34%
~99.9%
R = Recombinant NR = Nonrecombinant
Table 2. Analysis of recombination frequencies by plaque assays. Plaque assays were done using viral inoculum from wild-type high titer viral stock (A), and 5-day transfection supernatants from Sf9 cells co-transfected with either AcRP23.LacZ Baculovirus DNA and pVL1392-XylE plasmid DNA (B) or BaculoGold™ Baculovirus DNA and pVL1392-XylE plasmid DNA (C) on X-gal plates. After 7 days the plates were analyzed and the number of recombinant (R) (yellow in the presence of Catechol) versus non-recombinant (NR) (blue in the presence of β-gal) plaques were noted above. Recombination frequencies were determined by the number of R versus NR plaques. Each lot of BaculoGold™ Baculovirus DNA undergoes testing to insure that the recombination efficiency is greater than 99%.
To improve selection and screening methods, a polyhedrin-driven lacZ gene coding for β-galactosidase was inserted into the virus genome. Preparation of linearized BaculoGold™ DNA removes the lacZ gene. Non-recombinant, lacZ positive plaques stain blue and recombinant, lacZ negative plaques are colorless. Recombinant virus are selected as colorless in a plaque assay overlayed with agar containing X-gal: (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).
7
AcNPV C6 Wild-type Baculovirus DNA The wild-type AcNPV DNA is a super-coiled, double-stranded, circular DNA molecule with a molecular weight of 130 kb BaculoGold™ DNA, AcRP23.lacZ DNA and AcUW1.lacZ DNA are all derivatives of the AcNPV wild-type DNA. Originally, AcNPV wild-type DNA was widely used for co-transfection with recombinant Baculovirus Transfer Vectors to obtain recombinant BV particles. However, the identification of recombinants is time-consuming, requires considerable skills, and the recombination frequency is only 0.1%. Wild-type AcNPV DNA has no advantages over BaculoGold™, AcRP23 DNA or AcUW1.lacZ DNA. PharMingen sells purified ready-to-use AcNPV C6 Wild-type Baculovirus DNA (Cat. No. 21103D).
BaculoGold™ Linearized Baculovirus DNA BaculoGold™ DNA22, 23 is a modified AcNPV Baculovirus DNA which contains a lethal deletion and does not code for viable virus (Fig. 2). Co-transfection of the BaculoGold™ DNA with a complementing Baculovirus Transfer Vector rescues the lethal deletion by homologous recombination. Since only the recombinant BaculoGold™ produces viable virus, recombination frequencies exceed 99%. The flanking sequences of the complementing vector’s promoter region must be derived from the polyhedrin locus of the AcNPV wild-type DNA. p10 locus derived vectors (pAcUW1, pAcUW41, pAcUW42, pAcUW43) will not recover the lethal deletion of BaculoGold™. Furthermore, not all polyhedrin derived vectors are compatible with BaculoGold™ DNA. The lethal deletion in BaculoGold™ spans 1.7 kb downstream of the polyhedrin gene. Small streamlined vectors may not contain the entire region and will not rescue the lethal deletion. PharMingen sells purified ready-to-use linearized BaculoGold™ DNA (Cat. No. 21100D).
AcNPV wt DNA polyhedrin gene
p10 gene
Bsu36I
Bsu36I
ORF603
LacZ
Bsu36I
Uncut BaculoGold™ DNA ORF1629
polyhedrin promoter
∆ORF603
∆LacZ
∆ORF1629
Cleaved BaculoGold™ DNA containing lethal deletion
Figure 2. Design of AcNPV BaculoGold™ DNA. The polyhedrin gene locus of AcNPV DNA has been altered in the following ways: (1) a lacZ gene has replaced the viral polyhedrin gene; (2) three Bsu36I cutting sites have been added, in ORF 603, 1629 and in lacZ, which do not alter the amino acid sequences of their coding regions. The modified AcNPV DNA is linearized at the Bsu36I cutting sites deleting essential portions of the ORF 1629.
8
Linearized AcRP23.lacZ Baculovirus DNA Linearized AcRP23.lacZ DNA is a modified AcNPV Baculovirus DNA in which the viral polyhedrin gene was replaced by a lacZ gene (Fig. 3). AcRP23.lacZ is linearized at a single Bsu36I site introduced downstream of the polyhedrin promoter. Homologous recombination occurs during co-transfection of polyhedrin locus derived Baculovirus Transfer Vectors with AcRP23.lacZ. Approximately 30% of the resulting virus will be homologously recombined Baculovirus DNA. Since recombination disables the lacZ gene, recombinant Baculoviruses can be selected by plaque assay on X-gal plates. Nonrecombinant virus express the lacZ gene and plaques will appear blue. Recombinant virus do not express lacZ and plaques appear colorless. PharMingen sells purified readyto-use linearized AcRP23.lacZ DNA (Cat. No. 21101D). Note: When using AcRP23.lacZ DNA, a plaque assay is necessary to identify and isolate recombinants from non-recombinant Baculovirus.
AcNPV wt DNA polyhedrin gene
p10 gene
Bsu36I
Uncut AcRP23.lacZ DNA ORF603
LacZ
ORF1629
polyhedrin promoter
Linearized AcRP23.lacZ DNA ORF603
LacZ
ORF1629
polyhedrin promoter
Figure 3. Design of AcRP23.lacZ DNA. The polyhedrin gene locus of AcNPV DNA has been altered in the following ways: (1) a lacZ gene has replaced the viral polyhedrin gene; (2) a single Bsu36I cutting site has been added downstream of the polyhedrin promoter. The modified AcNPV DNA is linearized at the Bsu36I site.
9
Linearized AcUW1.lacZ Baculovirus DNA Linearized AcUW1.lacZ DNA is a modified AcNPV Baculovirus DNA which contains a p10 promoter driven lacZ gene (Fig. 4). AcUW1.lacZ DNA is linearized at a single single Bsu36I introduced downstream of the p10 promoter. Homologous recombination occurs during co-transfection of p10 locus derived Baculovirus Transfer Vectors with AcUW1.lacZ. Approximately 30% of the resulting virus will be homologously recombined Baculovirus DNA. Since recombination disables the lacZ gene, recombinant AcUW1.lacZ DNA can be color-selected by plaque assay on X-gal plates. Non-recombinant virus express the lacZ gene and plaques appear blue. Recombinant virus do not express the lacZ gene and plaques appear colorless. Both non-recombinant and recombinant virus are occlusion body positive. PharMingen sells purified ready-to-use linearized AcUW1.lacZ DNA (Cat. No. 21102D). Note: When using AcUW1.lacZ DNA, a plaque assay is necessary to identify and isolate recombinants from non-recombinant Baculovirus.
AcNPV wt DNA polyhedrin gene
p10 gene
Bsu36I Uncut AcUW1.lacZ DNA p26
LacZ
p74
LacZ
p74
p10 promoter
Linearized AcUW1.lacZ DNA p26 p10 promoter
Figure 4. Design of AcUW1.lacZ DNA. The p10 gene locus of AcNPV DNA has been altered in the following way: (1) a lacZ gene has replaced the viral p10 gene; (2) a single Bsu36I cutting site was added downstream of the p10 promoter. The modified AcNPV DNA is linearized at the single Bsu36I cutting site.
10
4
General Methods
The steps necessary to construct recombinant Baculoviruses using BaculoGold™ DNA (Cat. No. 21100D) are outlined in Figure 5. Protocols for each step are given within this chapter.
BaculoGold™ DNA
Clone foreign gene into transfer vector Propagate and purify vector containing foreign genes Co-transfect into insect cells Amplify recombinant virus Produce recombinant protein Purify recombinant protein
Figure 5. Experimental scheme using BEVS. Choose the appropriate transfer vector and clone in the foreign gene. Propagate the transfer vector containing the foreign gene using competent cells and purify by suitable means. Co-transfect BaculoGold™ DNA and recombinant transfer vector into Sf9 insect cells. Amplify the resultant recombinant virus in Sf9 insect cells. Use the amplified viral stock to produce protein. Purify your protein using appropriate methods.
4.1 Selecting an Appropriate Baculovirus Transfer Vector The BaculoGold™ Starter Package and Transfection Kit (Cat. No. 21001K and No. 21100K) both contain the Transfer Vector Set pVL1392/1393 (Cat. No. 21201P) (Appendix A). The pVL1392 and pVL1393 vectors are based on the polyhedrin locus and contain an extended MCS downstream of a polyhedrin promoter (Appendix E). These vectors have been used extensively to express a variety of proteins and should be adequate in most cases (Chapter 4.6). However, your protein expression needs may require that you use a specialized vector. For this reason, a variety of different Baculovirus Transfer Vectors have been constructed. The choice of vector will be determined by the application of the purified recombinant protein and in some cases by the nature of the protein itself. This section is intended as a guide to help researchers choose a vector which best fits their needs. First, decide whether to clone the gene of interest into a Baculovirus Transfer Vector that will produce the authentic protein encoded by its own ATG, or into a fusion-protein vector providing an N-terminal tag. The BEVS allows the expression of full length authentic proteins and does not require the expression of an N-terminal fusion sequence. This is a major advantage over many other expression systems, although, for certain applications it may be desirable to express a fusion protein. The tag may provide a sequence which can be used to label or modify the protein in a desired way that may not be available with the
11
authentic protein. The pAcGP67 and pAcSecG2T vectors incorporate a secretion signal sequence fused to the desired protein to force the recombinant protein into the secretory pathway. A fusion tag may also ease purification of non-secreted proteins. The pAcGHLT and pAcHLT vectors contain GST and 6xHis tags which can be purified on glutathione and Ni-NTA Agarose beads, respectively. Secondly, a suitable promoter must also be chosen. Baculovirus encoded promoters can be divided into the following classes according to the time, the viral infection cycle and conditions under which they are activated. PharMingen’s vectors contain either late or very late promoters. Immediate Early Promoters: Baculovirus promoters, activated due to the action of insect encoded transcription factors, control early viral transcription factors. Early Promoters: Baculovirus promoters, activated before viral DNA synthesis occurs, usually control genes necessary for the onset of viral replication (not usually used for foreign gene expression). Late Promoters: Baculovirus promoters, active during and after viral DNA synthesis, when the cell is producing Baculovirus components, control genes necessary to assemble the virus particles (e.g., 39K protein promoter, basic protein promoter). Very Late Promoters: Baculovirus promoters, activated very late during the infection cycle, well after virion assembly has been completed, control genes involved in the formation of occlusion bodies and cell lysis. Most genes controlled by very late promoters are non-essential under tissue culture conditions (e.g., p10 promoter, polyhedrin promoter). The early and immediate early promoters are generally very weak and are not routinely used in Baculovirus Transfer Vectors. The late promoters (the 39K and basic protein promoters) are moderately strong promoters which express their products late in the infection cycle when enzymes needed for post-translationally modified proteins are still present. The pAcMP2 and pAcMP3 transfer vectors (Cat. No. 21209P) contain the basic protein promoter and should be considered when the foreign protein is glycosylated, phosphorylated, etc. The polyhedrin and the p10 protein promoters are very strong promoters expressed during the very late phase of viral infection. They are essentially non-competitive and have been used together to construct multiple promoter vectors. The polyhedrin promoter is most commonly used and has been cloned into a variety of Baculovirus Transfer Vectors. Third, consider whether you want to use a single or multiple promoter vector. Multiple promoter vectors are useful for expressing subunits of heterodimers or for expressing a cell type- or tissue type-specific modifying enzyme along with your protein of interest. Table 3 is designed to help you to decide which Baculovirus Transfer Vector may be most appropriate for your work.
12
NA
A A A eD DN DN DN typ ld lacZ lacZ ildo w . . loG P23 W1 PV cu Ba AcR AcU AcN ™
Vector
Compatibility
Promoter
Type
Fusion Protein
Features
Cat. #
Polyhedrin locus-based Single Promoter Plasmids
pVL1392/3 (set) pAcSG2 pAcMP2/3 (set) pAcUW21 pAcGHLT-A, -B, -C (set) pAcHLT-A, -B, -C (set) pAcG1 pAcG2T pAcG3X BioColors™ BV Control (set) BioColors™ His (set)
• • • • • • • • • • •
• • • • • • • • • • •
• • • • • • • • • • •
Polyhedrin Polyhedrin Basic protein p10 Polyhedrin Polyhedrin Polyhedrin Polyhedrin Polyhedrin Polyhedrin Polyhedrin
very late very late late very late very late very late very late very late very late very late very late
no Standard polyhedrin locus vectors site dependent Recommended for large inserts, has an ATG no Facilitates post-translational modifications no Allows for in-larval expression, F1 origin yes GST-tag, 6xHis-tag thrombin cleavage site yes 6xHis-tag, thrombin cleavage site yes GST-tag yes GST-tag, thrombin cleavage site yes GST-tag, factor Xa cleavage site yes BioColors™ Genes yes BioColors™ Genes, 6xHis tag, thrombin cleavage site
21201P 21410P 21209P 21206P 21463P 21467P 21413P 21414P 21415P 21518P 21522P
• •
• •
• •
Polyhedrin Polyhedrin
very late very late
yes yes
Signal sequence Signal sequence, GST-tag
21223P 21469P
pAcUW51
•
•
•
Polyhedrin, p10 very late
no
21205P
pAcDB3
•
•
•
Polyhedrin, p10 very late
no
pAcAB3 pAcAB4 p10 locus-based
• •
• •
• •
Polyhedrin, p10 very late Polyhedrin, p10 very late
no no
Simultaneous expression of 2 foreign genes; F1 origin Simultaneous expression of 3 foreign genes; F1 origin Simultaneous expression of 3 foreign genes Simultaneous expression of 4 foreign genes
21216P 21412P
• •
p10
very late
no
Standard p10 locus vectors
21203P
• •
Polyhedrin, p10 very late
no
Simultaneous expression of 2 foreign genes; 21208P F1 origin
Secretory
pAcGP67 A, B, C (set) pAcSecG2T Multiple Promoter Plasmids
21532P
Single Promoter Plasmids
pAcUW1 Multiple Promoter Plasmids
pAcUW42/43 (pair)
13
Table 3. Vector Selection. The Vector Selection Chart gives a comprehensive overview of the vectors available for use with the BEVS. Please refer to Appendix E for vector maps and descriptions.
4.2 Optimizing Gene Expression Once the vector is chosen, the gene of interest is cloned into a restriction enzyme site downstream of the BV promoter. The efficiency of heterologous gene expression in the BV System can differ by approximately 1000 fold due to the intrinsic nature of the gene and the encoded protein. Modifying the heterologous gene will generally influence gene expression by only 2-5 fold. Researchers should not feel compelled to excessively modify their gene. However, there are some general rules for improving gene expression. Since translation will start at the first ATG initiation codon downstream of the chosen BV promoter, there should be no additional ATG codons upstream of the gene. Additionally, the 5’ untranslated sequence between the promoter and the start ATG should be kept to a minimum. In some cases, genes have been efficiently expressed from constructs with around 150 nucleotides between the promoter and the start ATG. However, it is advisable to trim down 5’ untranslated sequences to less than 50 nucleotides. The 3’ untranslated region downstream of the stop codon is of minor importance. There have been conflicting results regarding the importance of the polyadenylation signal. We have found that the expression level is generally not affected by the sequence downstream of the stop codons.
4.3 Cloning your Gene into a Baculovirus Transfer Vector The techniques required for inserting a foreign sequence into a Baculovirus Transfer Vector and preparing high quality plasmid DNA for co-transfections are described in this chapter. Most of the techniques are not unique to BEVS and we suggest referring to molecular biology manuals for supplementary cloning information.24, 25
Preparing Vector and Insert Examine the endonuclease restriction map for both the transfer vector and your gene of interest. Identify restriction site(s) common to the cloning site of the vector and to your gene of interest. The 5’ cloning site of your insert should be as close as possible to the ATG start codon of your gene (not more than 100 bases upstream). A polyadenylation sequence for the 3’ cloning site is optional and has not been shown in this system to improve stability or expression of recombinant protein. Both the insert and Baculovirus Transfer Vector DNA should be digested with appropriate restriction enzymes to generate compatible ends for cloning. If a single restriction enzyme is used to prepare the vector, the DNA must be treated with calf intestinal alkaline phosphatase (CIAP) to remove 5’ phosphate groups and prevent recirculation of the vector during ligation. When preparing the insert DNA, the correct restriction fragment (gene of choice) should be purified from an agarose gel by electroelution or DNA purification using glass-milk beads. PCR products should be similarly purified.
14
✓
Materials Needed
Agarose minigel (agarose concentration depends on the size range of the fragments) 0.5 M EDTA TE-saturated phenol/chloroform Chloroform:isoamyl alcohol (24:1) 7.5 M ammonium acetate Ethanol (100% and 70%) TBE gel electrophoresis buffer CIAP [(0.01 U/pmol of ends) if vector has been digested w/single endonuclease] TE buffer
1.
Prepare insert and Baculovirus Transfer Vector DNA by restriction endonuclease digestion. The following 20 µl reaction is provided as an example: 5 µl 1 µl 2 µl 12 µl 20 µl
plasmid DNA (1 µg/µl) appropriate restriction enzyme (e.g. BamHI, 20 U/µl) appropriate restriction buffer (10X) sterile deionized water final volume
2.
Incubate sample(s) at the appropriate temperature (depending on the restriction endonuclease used, usually 37°C) for 2–4 h.
3.
If the vector has been digested with a single restriction endonuclease, the DNA should be treated with CIAP. Thus, add the following components directly to the restriction endonuclease digest after the incubation time has been completed: 20 µl 3 µl 1 µl 6 µl 30 µl
previous volume CIAP 10X buffer CIAP sterile deionized water new final volume
4.
Incubate for 20 min at 37°C.
5.
Add 1 volume of TE-saturated phenol/chloroform. Vortex each sample for 10 s and centrifuge samples for 5 min at 12,000 × g in a microcentrifuge.
6.
Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloroform:isoamyl alcohol (24:1). Vortex each sample for 10 s and centrifuge samples for 2 min at 12,000 × g in a microcentrifuge.
7.
Transfer the upper aqueous phase to a fresh tube and add 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ice-cold 100% ethanol. Mix carefully by slowly inverting tubes several times by hand. Precipitate DNA by placing for 1 h at –20°C or 20 min on dry ice.
8.
Collect the DNA pellets by centrifugation at 12,000 × g for 5 min.
9.
Carefully remove the supernatant, wash the pellet with 1 ml of 70% ethanol, dry briefly in a 37°C oven or in a vacuum desiccator. Resuspend pellet in 20 µl TE buffer. Determine the approximate DNA concentration by agarose gel electrophoresis with comparison to known amounts of DNA standards.
15
Ligating Vector and Insert An insert DNA:vector molar ratio of 1:3, 1:1 and 3:1 should be used to determine optimal insert:vector ratios. The total amount of DNA for recessive-end cloning per 10 µl volume should be 200 ng. assuming:
si sv riv t i v
is the size of the insert is the size of the vector is the molar ratio of insert:vector is the amount of total DNA (insert plus vector) is the amount of insert needed in the DNA ligation reaction is the amount of vector needed in the DNA ligation reaction
the formula for this is as follows: i=
✓
(si x t) [(sv/riv) + si ]
and
v=
t x sv sv+ (si x riv)
Materials Needed
T4 DNA ligase 10X ligase buffer containing 10 mM ATP Sterile deionized water
1.
Set up a ligation reaction as described below. This example assumes an insert:vector ratio of 3:1.
Therefore, riv = 3. We define sv = 10 kb and si = 3.3 kb. The total DNA for recessive end cloning should be 200 ng. Therefore, t = 200 ng. If we insert these values into the formula above we calculate that we need 100 ng of vector DNA and 100 ng of insert DNA. Thus, our sample ligation looks as follows:
2. 3.
1 µl vector DNA (100 ng/µl, 10 kb) 1 µl insert DNA (100 ng/µl, 3.3 kb) 1 µl T4 DNA ligase (1 Weiss unit) 1 µl 10X ligase buffer 6 µl sterile deionized water 10 µl final volume Incubate the mixture at 16°C overnight. Following the ligation reaction, transform the ligated plasmid DNA (usually 1 µl of the ligation mixture) into competent cells of an appropriate host strain (e.g., HB101, DH5α). Note: To monitor the efficiency of the ligation and transformation steps, competent cells should also be transformed with uncut nonrecombinant vector DNA as well as cut vector DNA which has been ligated in the absence of an insert.
16
Propagating Vectors There are many different E. coli strains available which are suitable for preparation of competent cells used in transformations, e.g., DH5α or HB101. Many of these strains are available as commercially prepared competent cells. Several comprehensive manuals containing procedures for preparation of competent cells are listed in the Reference section of this manual. PharMingen’s transfer vectors are “high copy number” vectors and should generate yields of up to several milligrams per liter.
Transforming bacterial strains
✓
Materials Needed
SOB Medium (liter): 20 g Bactotryptone 5 g yeast extract 0.5 g NaCl Autoclave solution 2 M Mg solution Mix equal volumes of 2 M MgCl2 and 2 M MgSO4 Filter sterilize solution 2 M Glucose Filter sterilize solution LB/Amp (150 µg/ml) plates Competent cells β-mercaptoethanol
Before starting: Place DNA and 5 ml culture tubes on ice. Place culture plates in 37°C incubator to dry. Make SOC medium: (1 ml for each transformation) to each ml of SOB medium, add: • 10 µl 2M Glucose solution and • 10 µl 2M Mg solution Place SOC medium in the 37°C water bath. Make β-mercaptoethanol dilution - 1:20 dilution in sterile water. 1.
Thaw competent cells on ice (100 µl/ transformation).
2.
Gently thaw cells by hand. Aliquot 100 µl into pre-chilled 15 ml polypropylene tubes (Falcon Cat. No. 2097).
3.
Add 1.7 µl of the fresh β-mercaptoethanol dilution to the 100 µl of bacteria, resulting in a final concentration of 25 mM. Gently swirl.
4.
Incubate on ice for 10 min, swirling every 2 min.
5.
Add 0.1-50 ng of recombinant plasmid DNA (1 µl) to cells and swirl gently. As a positive control, add 1 ng of pBR322 to another 100 µl of cells.
6.
Incubate on ice for 30 min. Heat shock at 42°C for 45-55 seconds (critical!). Return to ice for 2 min.
7.
Add 0.9 ml of SOC medium preheated to 37°C. Incubate at 37°C for 1 h, shaking at 225 RPM on an orbital shaker.
17
8.
Spin down bacteria using a table-top centrifuge at 10,000 × g for 5 min. Remove all media except for 100 µl. Resuspend bacteria in remaining 100 µl and spread thin on an LB-Amp plate.
9.
Incubate plates at 37°C overnight.
10. The next day, pick up several colonies for miniprep DNA isolation to confirm the presence of the recombinant plasmid. Perform restriction endonuclease analysis to confirm the presence and orientation of the insert. Note: After transformation of a suitable E. coli host strain (e.g., HB101, DH5α, etc.) by a Baculovirus Transfer Vector and plating the bacteria on selective medium, cells harboring recombinant plasmid DNA will grow into colonies. Since all current Baculovirus Transfer Vectors contain an ampicillin resistant gene, the selection should be done on LB plates containing 50 µg/ml ampicillin.
Purifying Vectors The quality of both the vector and viral DNA is critical for successful co-transfections. Sf cells are sensitive to some contaminant’s found in crude plasmid preparations, which cannot be removed by phenol/chloroform extraction or ethanol precipitation. Vector DNA purified by CsCl-EtBr density gradient centrifugation, anion exchange chromatography (QIAEX resin, QIAGEN Inc) or by extraction with glassmilk will be sufficiently pure for co-transfection. Refer to molecular biology manuals for comprehensive purification techniques.24, 25
4.4 Insect Cell Lines Several established insect cell lines are highly susceptible to AcNPV virus infection. The two most frequently used insect cell lines are Sf9 and Sf21 (Cat. No. 21300L and No. 21301L). Both cell lines were originally established from ovarian tissues of Spodoptera frugiperda larvae and are highly recommended for use in the BEVS. Healthy insect cells attach well to the bottom of the plate forming a monolayer and double every 18–24 h. Infected cells become uniformly round, enlarged, develop enlarged nuclei, don’t attach as well and stop dividing. Sf9 and Sf21 cells may also be grown in suspension. Antibiotics are not required, but gentamicin sulfate (50 µg/ml) and Amphotericin B (“Fungizone”) (2.5 µg/ml) are often added to the media. CO2 supplementation is not required. We routinely use Sf9 cells and will refer to them from here on; however, Sf21 cells may be substituted. We commonly receive questions concerning cell confluency and BEVS assays. Table 4 was designed to help new users determine accurate cell densities per desired assay. Assay
Plate Size
# Cells per Assay
% Confluent
# Cells/ Confluent Plate
Transfection
60 mm
2.0 x 106
~60%
3.2 x 106
Dilution Assay
12 well
1.0 x 105
~30%
3.0 x 105
Plaque Assay
100 mm
6.2 x 106
~70%
8.8 x 106
150 mm
2.0 x 10
7
~70%
2.9 x 107
2.0 x 10
7
~70%
2.9 x 107
Viral Amplification Protein Production
150 mm
Table 4. Recommended cell numbers and approximate densities for various assays. These numbers are routinely used for Sf9 insect cell cultures in PharMingen’s laboratories. Individual users may want to further optimize these numbers for their own experimental systems.
18
General Handling Techniques The following information is helpful when handling insect cells.
•
Healthy Sf9 cells generally double every 18–24 h when grown in TNM-FH media (Cat. No. 21227M).
•
To maintain healthy cultures, Sf9 cells should be subcultured 1:3 when they reach confluency on plates (three times a week). They will grow reasonably well at temperatures between 26-28°C. However, after infection it is important to keep the temperature at 27°C ±0.5°C; otherwise, recombinant protein production may be poor, although cells will look infected.
•
An adjustment period ranging from a few days to several weeks should be allowed when transferring Sf9 cells between monolayer and suspension cultures.
•
Always equilibrate insect cell culture medium to RT before using.
•
When removing liquid from a plate of cells, tip the flask at a 30–60° angle so the liquid pools toward the bottom of the flask. Remove the liquid without touching the cell monolayer using a sterile pipette.
•
When seeding cells into a tissue culture plate or flask, be sure the vessels are placed on a flat surface to ensure homogenous cell density.
•
It is extremely important when doing a plaque assay to provide the proper cell density for plaque formation (Table 4). Rock the plate back and forth to evenly distribute the cells over the surface of the plate.
•
To pellet cells, gently dislodge cells from monolayers and transfer the cell suspension to a sterile centrifuge tube of appropriate size. Spin the suspension for 2–5 min at 1,000 × g. Carefully remove the supernatant without disrupting the cell pellet. To resuspend the cell pellet for culture, add the desired volume of fresh medium to the side of the tube and gently resuspend the pellet by pipetting the suspension up and down several times.
•
Insect cells are sensitive to centrifugal forces. For resuspension, cells should be centrifuged for 2–5 min at 1,000 × g in a GH-3.7 Beckman GPR horizontal rotor or equivalent.
•
TNM-FH and Grace’s medium do not contain pH color indicators. These media usually have a pH around 6.2.
•
Cell viability may be checked using trypan blue. To 1 ml of cells add 0.1 ml of a 0.4% stock solution of trypan blue (in PBS or other isotonic salt solution). Non-viable cells will take up trypan blue. Healthy, log-phase cultures should contain more than 97% unstained viable cells.
•
To minimize centrifugation cells may be transferred to a new tissue culture plate using the old medium. Once cells have adhered (10 min), change to fresh media.
Insect cells grow well both in suspension and as monolayer cultures and can be transferred from one to the other with minimal adaptation (Fig. 6). Small-scale propagation of cells can be maintained on plates; however, for large scale it is time-consuming and costly to use plates. Spinner flasks are ideal for scaling up insect cell cultures. Both monolayer and suspension cultures should be evaluated for optimal levels of protein expression.
19
A
B
Figure 6. Monolayer and suspension Sf cultures. A) Monolayer cultures. 150 mm tissue culture plates (Falcon Cat. No. 3025) used at PharMingen for protein production. B) Suspension cultures. 2 L, 1 L and 5 L (not shown) spinner flasks (Techne) used at PharMingen for cell propagation. Sf9 cells are suspended in Techne spinner flasks by a magnetic arm that spins at ~60 rpm. The culture volume should always remain less than half of the full volume of the flask. For example, a 1-liter flask should contain
