Baculovirus%20expression%20system

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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

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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

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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.

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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

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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)

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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.

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Figure
Tn7R p10
Gent Tn7L
Gene of Interest
Gene construct
Gene of Interest
PpH
Tn7 L
Tn7 R
pfast Bac with insert
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Contd..
Tn7R GOI Tn7L
Transposed pfast Bac
Bacmid DNA
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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 2300bpsize of insert
• Recombinant bacmid is now ready to transfect to
  insect cell lines

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Insect Medium

• Graces Insect medium- unsupplemented but
  contains L-glutamine
• Graces Insect medium supplemented-contains
  additional TC yeastolate Lactalbumin
  hydrolysate
• Trichoplusia ni Medium formulation hink (TNM-FH)-
  contains 10 FBS

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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

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Seeding density of 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
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Density 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 106
75 1.2 x 107 1.1 x 107 9.0 x 106
150 2.4 x 107 2.3 x 107 1.8 x 107
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Types of cell culturing

• Monolayer culture
• Suspension culture

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Methods of sub culturing adherent cells

• Three methods to dislodge monolayers in adherent
  cell culture
• - Sloughing
• -Trypsinization
• -Tapping the layer until monolayer loosens

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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

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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

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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

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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

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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

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Types of Insect cell lines
cells 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 five SFM Ovarian cells of cabbage looper Grow well as monolayer, also as suspension
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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

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Cryopreseravtion of cells

• Freezing cells should be 90 viable and
  80-90confluent
• Freezing medium should have 60 Graces insect
  medium supplemented with 30FBS 10 DMSO

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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

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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

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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

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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
  15 dilution

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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

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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