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Baculovirus expression system

Baculovirus expression system. Paras Yadav 1 , Annu Yadav 1 , P. Kumar 1 , J.S. Arora 1 , T.K.Datta 1 , S. De 1 , S.L. Goswami 1 , Mukesh Yadav 2 , Shalini Jain 3 , Ravinder Nagpal 4 and Hariom Yadav 3

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Baculovirus expression system

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  1. Baculovirus expression system Paras Yadav1, Annu Yadav1, P. Kumar1, J.S. Arora1, T.K.Datta1, S. De1, S.L. Goswami1, Mukesh Yadav2, Shalini Jain3, Ravinder Nagpal4 and Hariom Yadav3 1Department of Animal Biotechnology, 3Animal Biochemistry Division and 4Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana), India; 2SOS in Chemistry, Jiwaji University, Gwalior-474011, M.P., India

  2. Baculovirus • Baculovirus are present in invertebrates primarily insect species • They are not infectious for vertebrates & plants • Genome is covalently closed circular double stranded of 134 kbp, due to its small it can accommodate large fragments of foreign DNA • They are divided into two groups on the basis of their structure as-: Nucleopolyhedroviruses (NPV) Granuloviruses These NPV are mainly used as expression vectors i.e. Autographa californica NPV (AcMNPV) isolated from the larva of the alfalfa looper

  3. Contd.. • Baculovirus expression system based upon the ability to propagate AcMNPV in insect cells • Uses many of the protein modification, processing • and transport systems present in higher eukaryotic • cells. • Virus that can be propagated to high titers adapted • for growth in suspension cultures • obtain large amounts of recombinant protein with • relative ease • Baculovirus are noninfectious to vertebrates and • their promoters are inactive in mammalian cells.

  4. Insects & Insect cells • Baculovirus infects lepidopteran (butterflies & moths) insects and insect cell lines • Commonly used cell lines are sf9 & sf21 derived from the pupal ovarian tissue of the fall army worm spodoptera frugiperda and high five derived from the ovarian cells of the cabbage looper

  5. Baculovirus expression system • Recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insects larvae • Heterologous genes placed under the transcriptional control of the strong polyhedrin promoter of the Autographa californica polyhedrosis virus (AcNPV) • Based on site specific transposition of an expression cassette (pfast Bac with gene of interest) into a baculovirus shuttle vector (bacmid)

  6. Steps in recombinant baculovirus production • Clone the gene of interest in pfast Bac donor plasmid • Expression cassette in pfast Bac is flanked by left and right arms of Tn7 and also an SV40 polyadenylation signal to form a miniTn7 • Cloned pfast Bac is transformed in E.coli host strain (DH10Bac) which contains a baculovirus shuttle vector bacmid having a mini-attTn7 target site • Helper plasmid which allows to transpose the gene of interest from pfast to bacmid (shuttle vector) • Transposition occurs between the mini-att Tn7 target site to generate a recombinant bacmid • This recombinant bacmid can now be used to transfect insect cell lines.

  7. Figure… Tn7R p10 Gent+ Tn7L Gene of Interest Gene construct Gene of Interest PpH Tn7 L Tn7 R pfast Bac with insert

  8. 128bp 145bp Mini att Tn7 M 13 reverse M 13 forward Contd.. Tn7R GOI Tn7L Transposed pfast Bac Bacmid DNA

  9. Contd.. • PCR amplification using M-13 Forward and Reverse primers • If no transposition, then a region a bacmid alone will amplify to gave product of 300bp • In condition of transposition then the amplified size will be 2300bp+size of insert • Recombinant bacmid is now ready to transfect to insect cell lines

  10. Insect Medium • Grace’s Insect medium- unsupplemented but contains L-glutamine • Grace’s Insect medium supplemented-contains additional TC yeastolate & Lactalbumin hydrolysate • Trichoplusia ni Medium formulation hink (TNM-FH)- contains 10% FBS

  11. Requirements for proper cell culture • Temperature- Optimal range is 27-28 C • pH- Optimal range is 6.1 to 6.4 • Aeration-Requires passive 02 diffusion for optimal growth & recombinant protein expression • Osmolality- Optimum is 345-380 mOsm/kg • FBS- Working with suspension culture it is advisable to use (10-20% FBS) to gave protection from cellular shear forces

  12. Seeding density of cells

  13. Density of Insect Cells at confluency • Cell number may vary depending upon the culture conditions and the health of the culture

  14. Types of cell culturing • Monolayer culture • Suspension culture

  15. Methods of sub culturing adherent cells • Three methods to dislodge monolayers in adherent cell culture - Sloughing -Trypsinization -Tapping the layer until monolayer loosens

  16. Procedure of monolayer sub culture • Monolayer should reach to confluency in 2-4 days. • Serum supplemented cultures do not adhere to surface tightly where as serum free attach very tightly to substrates Aspirate medium & floating cells from a confluent monolayer & discard them. • Add 4ml of RT complete growth medium to each 25cm2 flask(12 ml to a 75 cm2 flask) • Resuspend cells by pipetting the medium across the monolayer with a Pasteur pipette. (Enzymatic dissociation is not recommended) • Observe cell monolayer using an inverted microscope to ensure adequate cell detachment

  17. Contd.. • Perform viable cells count on harvested cells. • Inoculate cells at 2 x 105 viable cells/ml into respective culture vessels. • Inoculate cultures kept at 25-28 C with loose caps to allow gaseous exchange • On day 4 post-planting, aspirate the spent medium from one side of the monolayer & subculture the flask • With slower growing cell lines, it may be necessary to feed the flasks on day 3-4 post planting • Subculture the flasks when the monolayer reaches 80-100% confluency, approx 2-3 days post planting

  18. Working with suspension culture • Insect cells are not generally anchorage dependent & can be well adapted to suspension culture • Prior to establish a spinner culture, cells are maintained firstly as healthy adherent cells. • Cell density reaches to 2-2.5 x 106 cells/mlthey should be diluted to no less than 7 x 105 cells/ml • Use a spinner flask with a vertical impeller • Culture volume should not exceed half of the volume of the flask • Use of surfactant to decrease shearing e.g. Pluronic F-68

  19. Contd.. • Not necessary to change medium regularly. Sub culturing requires the removal of cell suspension & the addition of medium • Impeller should be rotating regularly • Impeller should be submerged 1 cm or more to ensure adequate aeration • Cell viability of 95% is required • Minimum density of 1 x 106 cells/ml is required

  20. Contd… • Keep record of the passage number. After 30 passage or more (2-3 months), cells doubling time increased and also loose their viability and infectivity. • Keep a cell log, to do so one should have a knowledge of following; date of initiation of culture, lot number date of passage & passage number density & viability at passage comment on cell appearance medium & its lot number

  21. Types of Insect cell lines

  22. Initiation of culture with freezed cells • Thaw the frozen suspension rapidly in a water bath at 28 C • Seed the cells into a culture flask (1 x 106) containing medium 5 ml TC 100 medium • Incubate at 28 C for 5 hrs • Change with fresh medium • Incubate again, until it reach confluence • Subculture it for experimental purpose

  23. Cryopreseravtion of cells • Freezing cells should be 90% viable and 80-90%confluent • Freezing medium should have 60% Grace’s insect medium supplemented with 30%FBS & 10% DMSO

  24. Procedure • Count cells using haemocytometer • Placed cryovials on ice & label them • Centrifuge cells at 400-600 g for 10 mts at RT. Remove the supernatant • Resuspend the cells to the given density in the freezing medium • Transfer 1 ml of the cell suspension to sterile cryovials • Place at -20 C for 1 hr then transfer to -80 C for 24-48 hrs & then finally store at Liquid nitrogen

  25. Advantages of working with Baclo system • High expression levels using the polyhedrin or p10 promoter • Supports post-translation modifications • BEVS enables simultaneous expression of certain genes • Expressed proteins do not have size limitations • Capable of producing cytotoxic proteins

  26. Leukemia in working with BEVS • Baculovirus system works only in invertebrates so the expressed vertebrate proteins are different in post translation modifications with high mannose type glycosylation. • It has limited capacity to properly processed inactive precursor proteins due to the absence of pro-protein convertases • Limited protein yield due to accumulation of insoluble protein within the cells

  27. Do and Don'ts • Check cells daily until a confluent monolayer is formed. • Passage cells at confluency only, as cells will be easy to dislodge & shows better viability • Do not overgrow cells, it results in decreased viability • Do not splits cells too for. Densities lower than 20% confluency inhibit growth • Passage the cells only in log phase, log phase growth can be maintained by splitting cells in 1:5 dilution

  28. Basic aseptic conditions • If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen • Swab all bottle tops & necks with 70% ethanol • Flame all bottle necks & pipette by passing very quickly through the hottest part of the flame • Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger • Work either left to right or vice versa, so that all material goes to one side, once finished • Clean up spills immediately & always leave the work place neat & tidy

  29. Contd.. • Possibly keep cultures free of antibiotics in order to be able to recognize the contamination • Never use the same media bottle for different Insect cell lines. If caps are dropped or bottles touched unconditionally touched, replace them with new ones • Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200 C • Switch on the laminar flow cabinet 20 mts prior to start working • Cell cultures which are frequently used should be subcultered & stored as duplicate strains

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