Baculovirus expression Baculovirus expression systemsystem

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

BaculovirusBaculovirus• 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

Contd..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.

Insects & Insect cellsInsects & 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

Baculovirus expression Baculovirus expression systemsystem

• 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)

Steps in recombinant Steps in recombinant baculovirus productionbaculovirus 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.

Figure…Figure…

Gene of Interest

Tn7R p10 Gent+ Tn7L

Gene construct

Gene of Interest

Tn7 R

PpH Tn7 L

pfast Bac with insert

Contd..Contd..

128bp 145bp

Mini att Tn7M 13 forward M 13 reverse

Tn7R GOI Tn7L

Bacmid DNA

Transposed pfast Bac

Contd..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

Insect MediumInsect 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

Requirements for proper cell Requirements for proper cell cultureculture

• 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

Seeding density Seeding density of cellsof cells

Disk/flask Seeding density(cell number)

Vol. of medium (ml)

35mm dia. Petri dish

1x 105 1.5-2

60mm dia. Petri dish

2 x 105 3-4

25 cm2 flask 1x 106 4-5

75 cm2 flask .5-1 x 107 10

Spinner culture 1-2 x 105/ml 40-500

Density of Insect Cells at confluencyDensity of Insect Cells at confluency

• Cell number may vary depending upon the culture conditions and the health of the culture

Flask size(cm2)

sf9 sf21 High five

25 4 x 106 3.8 x 106 3.0 x 10675 1.2 x 107 1.1 x 107 9.0 x 106150 2.4 x 107 2.3 x 107 1.8 x 107

Types of cell culturingTypes of cell culturing

• Monolayer culture

• Suspension culture

Methods of sub culturing Methods of sub culturing adherent cellsadherent cells

• Three methods to dislodge monolayers in adherent cell culture

- Sloughing -Trypsinization -Tapping the layer until monolayer loosens

Procedure of monolayer sub Procedure of monolayer sub cultureculture

• 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

Contd..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 cultureWorking 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..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…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 linesTypes of Insect cell linescells Doubling

time Cell appearance

Medium Origin Type of culture

Sf 9 72 hrs Spherical, granular, regular in size, firm attachment to surface

TNM-FH IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda

Grow well as monolayer and suspension

Sf 21 24 hrs Spherical, granular, different in size, firm attachment to surface

TNM-FH IPLBSF-21 cell lines of the fall army worm spodoptera frugiperda

Grow well as monolayer and suspension

High-five 18 hrs Spherical, granular, regular in size, loose attachment to surface

Express fiveSFM

Ovarian cells of cabbage looper

Grow well as monolayer, also as suspension

Initiation of culture with Initiation of culture with freezed cellsfreezed 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 Cryopreseravtion of cellscells

• 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

ProcedureProcedure• 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 Advantages of working with Baclo systemwith 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

Leukemia in working with Leukemia in working with BEVSBEVS

• 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'tsDo 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 conditionsBasic 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..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