Baculovirus expression system Paras Yadav 1 Annu Yadav

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Baculovirus expression system Paras Yadav 1, Annu Yadav 1, P. Kumar 1, J. S.

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 1 Department of Animal Biotechnology, 3 Animal Biochemistry Division and 4 Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana), India; 2 SOS in Chemistry, Jiwaji University, Gwalior-474011, M. P. , India

Baculovirus • • Baculovirus are present in invertebrates primarily insect species They are not

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 (Ac. MNPV) isolated from the larva of the alfalfa looper

Contd. . • • • Baculovirus expression system based upon the ability to propagate

Contd. . • • • Baculovirus expression system based upon the ability to propagate Ac. MNPV 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.

Insects & Insect cells • • Baculovirus infects lepidopteran (butterflies & moths) insects and

Insects & Insect cells • • Baculovirus infects lepidopteran (butterflies & moths) insects and insect cell lines Commonly used cell lines are sf 9 & sf 21 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

Baculovirus expression system • • • Recombinant baculovirus have become widely used as vectors

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 (Ac. NPV) Based on site specific transposition of an expression cassette (pfast Bac with gene of interest) into a baculovirus shuttle vector (bacmid)

Steps in recombinant baculovirus production • • • Clone the gene of interest in

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 Tn 7 and also an SV 40 polyadenylation signal to form a mini. Tn 7 Cloned pfast Bac is transformed in E. coli host strain (DH 10 Bac) which contains a baculovirus shuttle vector bacmid having a mini-att. Tn 7 target site Helper plasmid which allows to transpose the gene of interest from pfast to bacmid (shuttle vector) Transposition occurs between the mini-att Tn 7 target site to generate a recombinant bacmid This recombinant bacmid can now be used to transfect insect cell lines.

Figure… Tn 7 R p 10 Gent+ Gene of Interest Tn 7 L Gene

Figure… Tn 7 R p 10 Gent+ Gene of Interest Tn 7 L Gene construct Gene of Interest Pp. H Tn 7 L Tn 7 R pfast Bac with insert

Contd. . Tn 7 R GOI Tn 7 L Transposed pfast Bac 128 bp

Contd. . Tn 7 R GOI Tn 7 L Transposed pfast Bac 128 bp M 13 forward 145 bp Mini att Tn 7 Bacmid DNA M 13 reverse

Contd. . • • PCR amplification using M-13 Forward and Reverse primers If no

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 300 bp In condition of transposition the amplified size will be 2300 bp+size of insert Recombinant bacmid is now ready to transfect to insect cell lines

Insect Medium • • • Grace’s Insect medium- unsupplemented but contains L-glutamine Grace’s Insect

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 (TNMFH)- contains 10% FBS

Requirements for proper cell culture • • • Temperature- Optimal range is 27 -28

Requirements for proper cell culture • • • Temperature- Optimal range is 27 -28 C p. H- Optimal range is 6. 1 to 6. 4 Aeration-Requires passive 02 diffusion for optimal growth & recombinant protein expression Osmolality- Optimum is 345 -380 m. Osm/kg FBS- Working with suspension culture it is advisable to use (10 -20% FBS) to gave protection from cellular shear forces

Seeding density of cells Disk/flask Seeding density (cell number) Vol. of medium (ml) 35

Seeding density of cells Disk/flask Seeding density (cell number) Vol. of medium (ml) 35 mm dia. Petri dish 1 x 105 1. 5 -2 60 mm dia. Petri dish 2 x 105 3 -4 25 cm 2 flask 1 x 106 4 -5 75 cm 2 flask . 5 -1 x 107 10 Spinner culture 1 -2 x 105/ml 40 -500

Density of Insect Cells at confluency • Cell number may vary depending upon the

Density of Insect Cells at confluency • Cell number may vary depending upon the culture conditions and the health of the culture Flask size(cm 2) sf 9 sf 21 High five 25 4 x 106 3. 8 x 106 3. 0 x 106 75 1. 2 x 107 1. 1 x 107 9. 0 x 106 7 2. 3 x 107 1. 8 x 107 150 2. 4 x 10

Types of cell culturing • Monolayer culture • Suspension culture

Types of cell culturing • Monolayer culture • Suspension culture

Methods of sub culturing adherent cells • Three methods to dislodge monolayers in adherent

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

Procedure of monolayer sub culture • • • Monolayer should reach to confluency in

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 4 ml of RT complete growth medium to each 25 cm 2 flask(12 ml to a 75 cm 2 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

Contd. . • • • Perform viable cells count on harvested cells. Inoculate cells

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

Working with suspension culture • • • Insect cells are not generally anchorage dependent

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/ml they 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

Contd. . • • • Not necessary to change medium regularly. Sub culturing requires

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

Contd… • • Keep record of the passage number. After 30 passage or more

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

Types of Insect cell lines cells Doubling time Cell appearance Medium Origin Sf 9

Types of Insect cell lines cells Doubling time Cell appearance Medium Origin Sf 9 Type of culture 72 hrs Spherical, granular, regular in size, firm attachment to surface TNM-FH IPLBSF-21 cell Grow well as lines of the fall monolayer and army worm suspension spodoptera frugiperda Sf 21 24 hrs Spherical, granular, different in size, firm attachment to surface TNM-FH IPLBSF-21 cell Grow well as lines of the fall monolayer and army worm suspension spodoptera frugiperda High-five 18 hrs Spherical, granular, Express five Ovarian cells regular in size, loose SFM of cabbage attachment to looper surface Grow well as monolayer, also as suspension

Initiation of culture with freezed cells • • • Thaw the frozen suspension rapidly

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

Cryopreseravtion of cells • • Freezing cells should be 90% viable and 8090%confluent Freezing

Cryopreseravtion of cells • • Freezing cells should be 90% viable and 8090%confluent Freezing medium should have 60% Grace’s insect medium supplemented with 30%FBS & 10% DMSO

Procedure • • • Count cells using haemocytometer Placed cryovials on ice & label

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

Advantages of working with Baclo system • • • High expression levels using the

Advantages of working with Baclo system • • • High expression levels using the polyhedrin or p 10 promoter Supports post-translation modifications BEVS enables simultaneous expression of certain genes Expressed proteins do not have size limitations Capable of producing cytotoxic proteins

Leukemia in working with BEVS • • • Baculovirus system works only in invertebrates

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

Do and Don'ts • • • Check cells daily until a confluent monolayer is

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

Basic aseptic conditions • • • If working on the bench use a Bunsen

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

Contd. . • • • Possibly keep cultures free of antibiotics in order to

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