Learning to Express and Purify Mammalian Proteins

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Learning to Express and Purify Mammalian Proteins

• James L. Hartley, Ph.D. Advanced Technology
  ProgramSAIC-Frederick, Inc.National Cancer
  Institute at Frederick
• Cancer Biotechnology Lecture
• April 19, 2007

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Advice for those starting a protein expression
and purification project
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Suggestions

• ... when beginning a project
• Decide which version of the gene to attempt (many
  SNPs, splice variants).
• Buy an MGC clone (lt100) if possible, or trust
  but verify a laboratory clone.
• The literature may not be a good predictor of
  success. Read papers critically.
• Often the choice of method depends on the
  ultimate use of the protein
• How much, how pure (endotoxin?), how active?
• For structure, biochemistry, therapeutics,
  antigen?
• Fusion OK? Native at both ends?

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Suggestions

1. A good antibody can be very important for
   monitoring expression and purification (add an
   epitope if necessary).
2. Consider different expression clones up front
   (saves time if you have to change course).
3. If PCR of the gene is difficult, suspect the
   wrong sequence.
4. If the protein is cytoplasmic, insect cells with
   baculovirus are most likely to succeed (but E.
   coli is faster to test).
5. If resources permit, consider testing E. coli and
   insect cells in parallel.
6. For baculovirus expression, always test different
   conditions if yield and quality are important.

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Suggestions

• If a little active protein would be valuable,
  test samples that look completely insoluble.
• If the protein is secreted, many options are
  available.
• Mammalian cells can give high yields but take
  time and .
• Insect cells with baculovirus are faster for tens
  of mg.
• K. lactis or P. pastoris should be considered if
  fermentation is available.

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Suggestions

• If protein results are poor (low expression or
  low solubility or low activity), consider
• Related proteins (a single amino acid can make
  all the difference).
• Domains or fragments.
• Mutants.
• If the protein is a membrane protein, try for an
  extracellular domain first.
• For an integral membrane protein, be prepared for
  a long struggle, and have an activity assay.

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Introduction We make YOUR protein

• Protein Expression Laboratory
• Part of the Advanced Technologies Program,
  NCI-Frederick / SAIC-Frederick.
• Clone genes for protein expression.
• Express genes in E. coli, insect, mammalian,
  yeast cells.
• Purify proteins.
• Integrated projects (cloning, expression, and
  purification).
• Small individual projects.
• Many high-risk projects (virtually all protein
  expression projects) qualify for 50 lower cost
  (OD support) (NCI only).

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Protein Expression Laboratory

• Clone OptimizationDominic Esposito, Leslie
  Garvey, Chako Chiakiath
• Molecular FermentationTroy Taylor, John-Paul
  Denson, Andrew Waters
• Eukaryotic ExpressionButch Hopkins, Veronica
  Roberts, Cammi Bittner
• Protein PurificationBill Gillette, Peter Frank,
  Angelina KlineBurgess, Bill Bere
• Protein TechnologiesDeb Chatterjee, Kala
  Sitaraman, Stan Kaczmarczyk
• AdministrationJim Hartley, Marilyn Lyles

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Protein Expression Laboratory

• Clone Optimization Group
• Gene (PCR template)
• Clone with protease site, epitope tag, signal
  peptide, etc., sequence (gt400 last year)
• Clone for client use, deliver transfection-ready
  DNA
• Subclone into expression vectors
• Prepare and QC bacmid DNAs for baculo testing
• Prepare E. coli vectors
• E. coli optimization

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PEL Clone Optimization Group

• Clone Optimization Group

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PEL Clone Optimization Group
Clone Optimization Group
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PEL Clone Optimization Group
Clone Optimization Group E. coli optimization
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PEL Clone Optimization Group

• Protein expression vectors (all Gateway)
• Bacterial 105 vectors, mostly different fusion
  tags
• Mammalian 74 vectors, fusion tags, promoters
• Insect 42 vectors, for baculovirus and transient
  expression
• 18 lenti-, adeno-, and retroviral vectors with
  CMV, UbC, U6, LTR promoters
• 13 yeast vectors, for Saccharomyces, Pichia,
  Kluyveromyces
• Sent to 60 labs worldwide, now through AddGene

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PEL Clone Optimization Group
Comparison of mammalian promoters to improve
yields in mammalian cells (GFP reporter).
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Protein Expression Laboratory

• Molecular Fermentation Group
• Strain construction and optimization.
• Shake flask or fully-instrumented fermenters.
• E. coli induction or autoinduction.
• E. coli growths to OD 20 200.
• Yeasts P. pastoris, K. lactis to OD gt200 (also S.
  cerevisiae).
• Outstanding for secreted proteins.
• Analysis of expression by Coomassie-stained or
  Western gels.

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Protein Expression Laboratory
Molecular Fermentation Group
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PEL Molecular Fermentation Group

• Molecular Fermentation Group Autoinduction in
  fermentation tanks

BM 5h 5h 6h 6h 7h 7h 8h
8h 9h 9h 10h 10h 11h 11h
12h 12h 13h 13h 14h 14h 15h 15h
BM I S I S I
S I S I S I
S I S I S I
S I S I S
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PEL Molecular Fermentation Group
Molecular Fermentation Group

• Instrumented fermenters are becoming more
  valuable for control than for volume
• Dissolved oxygen
• pH
• Temperature
• Nutrients
• Induction of protein expression

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PEL Molecular Fermentation Group

• mCherry chosen for
• Brightness (16, EGFP 34)
• Fast maturation (t1/2 15 min, EGFP 65 min)
• Monomeric

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PEL Molecular Fermentation Group
New expression hosts are not easy to bring on line
Expression of active GM-CSF in Pichia pastoris
(Greiner)
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PEL Molecular Fermentation Group
New expression hosts Kluyveromyces lactis (yeast,
galactose induction)
Expression of active interferon (Hy3) in K.
lactis (Zoon)
Interferon in culture medium
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Protein Expression Laboratory

• Eukaryotic Expression Group Baculovirus
  expression
• Virus-like particle production
• Monitoring cell parameters
• Mammalian transfection (reagent cost reduced from
  150 to 0.08 per liter) and co-transfection
• Stable expression in mammalian cells

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PEL Eukaryotic Expression Group
MOI studies cut volume required for Ebola-based
VLP production from 40 L to 10 L
MOI 3
MOI 10
MOI 20
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PEL Eukaryotic Expression Group

• Cedex cell counting, sizing, and viability give
  quick indications of toxicity and infection

ControlHigh Five cells
ControlSf-9 cells
InfectedHigh Five cells
InfectedSf-9 cells
Increasing cell size ?
Increasing cell size ?
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PEL Eukaryotic Expression Group
Mixing DNAs prior to transfection reagent
improves mammalian coexpression.
Mixed DNAs PEI
Separate DNA-PEI complexes
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PEL Eukaryotic Expression Group
Eukaryotic Expression Group baculo time course
Determine viral titer
Amplify virus
Transfect bacmid into Sf9 cells
Sf9, 27º C
Harvest cells, supes, or both, 48 and 72 hours
Infect Sf9 and Hi5 cells
Hi5, 27º C
Hi5, 21º C
Prepare soluble and insoluble fractions
Coomassie-stained gels and / or westerns, with
appropriate controls
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PEL Eukaryotic Expression Group
Eukaryotic Expression Group baculo time course
Coomassie
Western
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PEL Eukaryotic Expression Group

• Stable expression of mammalian protein
• Project with NCI-Frederick/SAIC-Frederick, Inc.,
  Biopharmaceutical Development Program (BDP)
• Plasmid from Harvard Medical School
• Clone reconstructed in PEL (replaced ApR with
  CmR)
• New plasmid tested in three mammalian cell lines.
• CHO-S transfectants cloned, ten supes tested by
  BDP
• Best clone transferred to BDP, assayed at 3 mg/L
• BDP optimization in GMP environment, now at 200
  mg/L

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Protein Expression Laboratory

• Protein Purification Group
• Native or affinity purification.
• Cytoplasmic or secreted proteins.
• Tag removal with native N-terminus.
• Microgram to gram scale.
• Monoclonal antibodies from culture medium.
• Low endotoxin if required.
• Low-cost scouting to design optimal protocols.

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PEL Protein Purification Group
Protein Purification Group Tag removal
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PEL Protein Purification Group
Protein Purification Group many contaminants
are protein-specific
Purified from E. coli
Purified from insect cells
5 Rattray 6 Randazzo 7 Garboczi 8
Liu 9 Henry 10 Shen 11 Muegge
5 6 7 8 9 10 11
1 Kurian 2 Kimura 3 Nissley 4 Melenhorst
1 2 3 4
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PEL Protein Purification Group
Protein Purification Group when things go well
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PEL Protein Purification Group
Early TEV digest (tag removal) and SEC (for
monodispersity)
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PEL Protein Purification Group
Micro-scale scouting Phynexus
Column bed volume in P200 tip
Project 4365 Randazzo Protein Panel A- AGAP2,
91 kDa (4031-X2-8) Panel B- ASAP1, 123 kDA
(4031-X3-8) Panel C- ARAP1, 163 kDa (4031-X5-8)
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Some PEL Successes
Protein Use Investigator Publication
Leishmania Antigen, vaccine studies Seder
YpV Antigen for vaccine studies Amemiya Powell
HIV gag Antigen for vaccine studies Seder
KHSV ORF73 Antigen for epidemiology studies Whitby gt 30 publications
SirT's Biochemistry (active) Barrett Michishita, et al., Mol Biol Cell. 2005
Apobec3g Biochemistry (active) Pathak
UGRP1 and 2 Biochemistry (active) Kimura
TLR3 ecd Structure Segal, Davies Bell, et al., PNAS 2006
IFNARs 1 and 2 ecd's Biochemistry (active) Zoon
GM-CSF Biochemistry (active) Greiner
Alpha interferons Biochemistry (active) Zoon Schmeisser, et al., J Interferon Cytokine Res. 2006
Autotaxin Biochemistry (active) Liotta, Bandle
Topoisomerase 1 Biochemistry (active), structure Pommier
Agouti Biochemistry (active) Hearing
Grb2 Small molecule interactions Burke, Fisher Oishi, et al., Bioorg Med Chem. 2005
LSH Biochemistry, structure Muegge, Wlodawer
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Some PEL Successes
Product Use Investigator Publications
Virus-like particles (Ebola, Rift Valley, Lassa, Marburg) Vaccine studies Panchal
140 Traf2 and 3 mutant expression clones Biochemistry Brown
175 Folliculin expression clones Biochemistry Schmidt, Zbar, Linehan Baba, et al., PNAS 2006
46K vials of NCI-60 cell lines, ongoing support Biology NCI gt500 publications
Active cytokines from P. pastoris, K. lactis Biochemistry Zoon Esposito, et al., Protein Expr. Purific. 2005
E. coli cells, 40X higher yield of GRFT Anti-HIV Mori Giomarelli, et al., Protein Expr. Purific. 2006
Gateway vectors Protein expression gt60 worldwide AddGene
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Protein Expression Laboratory

• Protein Technologies Group (Deb Chatterjee)
• Protein arrays
• New tandem affinity purification (TAP) vectors
  and procedures for discovering protein-protein
  interactions conversion to OD-supported standard
  service due May-June 2007
• Affinity purification tag based on leucine
  zippers
• Three new solubility tags
• Mouse metastasis model (with Melinda
  Hollingshead, Steve Hughes, manuscript in
  preparation)

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PEL Protein Technologies Group

• Uses for Protein Microarrays
• Screening for antibodies
• Protein-protein interactions
• Protein-drug interactions
• Screening for enzyme substrates

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PEL Protein Technologies Group

• Approaches to Protein Arrays
• Purify proteins one-by-one
• Short shelf life
• Expensive
• Synthesize proteins in situ
• Much greater flexibility
• Takes advantage of DNA array technology
• Make and use

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PEL Protein Technologies Group
Prior technology
Gene of interest GST
LaBaer, et al., Science 305, 86, 2004
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PEL Protein Technologies Group
Tus protein binds to Ter sites with very high
affinity.
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PEL Protein Technologies Group

• PTG Improvements
• Patent pending
• Evaluation by three companies in progress
• Manuscript in preparation

Gene of interest Tus His6
A
C
B
Target DNA
Protein binds to plasmid encoding it
Add cell-free expression cocktail
Chatterjee, et al., 2006
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PEL Protein Technologies Group
Human proteins expressed as fusions with Tus-His6
Anti-His6
Human B2
Human B8
Human H1
Human H7
Human B1
Human A1
Human D12
Human G2
Arrays courtesy of D. Munroe and C. Baptista,
Laboratory of Molecular Technology, ATP
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PEL Technology Development

• POET (Pooled ORF Expression Technology)
• Standard high throughput approach for expressing
  proteins Test each gene expression, solubility,
  purification
• POET Pool dozens or hundreds of genes, subclone,
  express, and purify together, identify highly
  expressed, soluble, purifiable proteins by 2D-gel
  and mass spectrometry
• 2nd generation POET (in progress) mass
  spectrometry for both quanitation and
  identification

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PEL Technology Development
POET results from 688 C. elegans ORFs Note 2
success rate using standard approach.
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More advice
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Genes for protein expression

• Add small fusion tags, translation signals,
  secretion signals, remove or change stops, with
  PCR (Phusion from NEB) (Esposito).
• Always sequence genes following PCR (errors in
  oligos).
• Worst source of genes other investigators, even
  if they have been used in the lab for years.
• Best source of genes cDNAs from Mammalian Gene
  Collection. Vendor of choice Open BioSystems.

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Genes for protein expression

• Stop codons should be TAA, at least in E. coli
  (TGA ? 10 readthrough in BL21(DE3)).
• For secretion from insect cells the Gp64/67
  signal peptide is better (in limited tests) than
  native mammalian signal peptides. Note that this
  signal peptide has been corrected and is now
  shorter (Novagen, NCBI).
• Can cytoplasmic proteins be secreted? Not in our
  tests (N5) (see also Beckwith).

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Observation

• Viruses have evolved to bypass limits to protein
  overexpression ? Elements from viruses are most
  likely to help us get high levels of expression.
• T7 RNAp in E. coli (pET vectors, BL21(DE3).
• Baculovirus for insect cells.
• CMV promoter in mammalian cells.
• IRES elements in mammalian cells.
• Reference J. Alwine NIH seminar November 16,
  2005

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Cloning for protein expression

• Use REaL (restriction enzymes and ligase) vectors
  if time is cheap and vectors are not too big.
• Use recombinational cloning (Gateway (Invitrogen)
  or Creator (Takara)) for best reliability and
  speed.
• Consider initial cloning into non-expression
  vector for sequencing, and to avoid toxicity.
• In-Fusion cloning (Takara) is an enzyme
  necessary (Bayley) ?
• Making good vector preps is not easy, for REaL,
  LIC, In-Fusion.
• Making good vectors is easier with a deadly
  selectable marker.

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Cloning for protein expression

• Use vectors to supply promoters, large fusions,
  drug markers, etc.
• Try AddGene for vectors for fewer .

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Cloning for protein expression

• E. coli expression vectors
• Usually low copy number (pBR ori) for reduced
  chance of toxicity (not good for sequencing or
  mutagenesis).
• Most are derived from pET vectors, phage T7
  promoter.
• His6 affinity tag combined with solubility tags
  such as MBP or NusA works best in E. coli (but
  mammalian proteins often behave badly when these
  are removed).

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Cloning for protein expression

• Insect cell / baculovirus expression vectors
• For high levels of protein use polyhedrin
  promoter.
• Suspect that weaker promoters (P10, basic) might
  be better when activity or solubility are poor.
• Transient expression with plasmids gives much
  lower yield than baculovirus (multiple viral
  proteins needed in trans for high level
  expression with viral promoters).
• MBP also improves solubility in insect cells.
• In contrast to E. coli expression, the GST fusion
  tag can improve solubility.

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Cloning for protein expression
In contrast to our experience in E. coli, GST can
be much better than MBP as an expression tag in
insect cells.
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Cloning for protein expression

• Mammalian expression vectors
• CMV promoter.
• Many other elements touted, none widely adopted,
  no side-by-side tests of many elements with
  multiple genes.
• Plasmids containing EBV oriP usually improve
  transient expression in 293E cells for us (Ren,
  2006)
• Introns (Promega pdf) sometimes help (but not
  always).

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Cloning for protein expression
Comparison of mammalian promoters to improve
yields in mammalian cells (GFP reporter).
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Cloning for protein expression

• Mammalian expression vectors
• Boundary / insulating / opening elements
• mRNA export elements (e.g., WRE) Zufferey et al.
  (cited many times, vector available from
  AddGene).
• Genes with the drug resistance on the same mRNA
  (IRES) are less likely to be silenced.

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Cells for protein expression

• E. coli cells
• Very far evolutionarily from mammalian cells.
• Lower success rate as size of protein increases
  (30-40 kDa).
• Use codon-optimized strains (compensate for
  mammalian codons that are rare in E. coli).
• A rare-codon version of Fred Blattners reduced
  genome strain should be tested.

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Cells for protein expression

• E. coli cells
• Studiers monograph on growth of E. coli is must
  reading.
• No (or very limited) post-translational
  modifications (glycosylation, phosphorylation,
  etc.) preferred for structural studies.
• Overexpressing foreign proteins is stressful (our
  POET results).

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Cells for protein expression

• Yeast cells
• Pichia pastoris grows to much higher densities
  (ODs gt200) than S. cerevisiae, in defined medium.
• Kluyveromyces lactis, a food yeast, is similar
  but licensing (from NEBioLabs) is much more
  straightforward (Pichia is split between RCT and
  Invitrogen).
• Fermenters required.
• Non-mammalian glycosylation (but also true of
  bacteria and insects even CHO cells are rodent).

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Cells for protein expression

• Insect cells with baculovirus
• Much closer to mammals than bacteria or yeast.
• Transient expression with plasmids give lower
  yields (multiple viral gene products needed for
  high level expression).
• Viral system host cells die, cells are harvested
  before lysis. Better for toxic proteins?
• Viral system bypasses limits to expression.
• Safety.
• Post-translational modifications useful
  phosphorylation has been reported (Google search
  for phosphorylation in insect cells)
• Glycosylation is not mammalian (Jarvis).

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Cells for protein expression

• Mammalian cells
• The obvious choice for most native
  glycosylation, post-translational modifications.
• Yields for cytoplasmic expression are low.
• Yields for secreted proteins can be very high
  (gt1g/L Butler) .
• Almost all pharmaceuticals made in mammalian
  cells are made in rodent cells (Schmidt).
• HEK293 (human embryonic kidney) may be neuronal
  (according to their originator Graham)

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Protein expression

• Expression in E. coli
• Grow at 37º, induce expression at lower
  temperature (we use 16º overnight) for improved
  solubility.
• Lower temps slow T7 RNAP and increase coupling
  between transcription and translation (Nierhaus).
• Note it takes a long time (3 hours) for a 1
  liter culture in a flask at 37º to cool to 16º in
  an air shaker (and to warm to 37º).

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Protein expression

• Expression in insect cells
• The problem step is inserting your gene into the
  baculovirus genome (134 kb).
• Bac-to-Bac (purify bacmids from E. coli
  Invitrogen).
• Transfect with PEI (polyethyleneimine). Form
  complexes in water or saline, not medium.
• Disposable Fernbach flasks (1 L capacity) for
  production.
• Coinfection for coexpression of multiple genes
  requires high mois.

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Protein expression
MOI 3
MOI 10
MOI 20
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Protein expression

• Expression in insect cells
• Smaller scale, higher throughput methods can be
  useful for side-by-side comparisons of multiple
  genes.
• For choosing conditions for expression i) titer
  virus ii) compare Sf9/27º, Hi5/27º, and Hi5/21º
  at 48 and 72 h.

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Protein expression
Example of insect cell time course appropriate
standards are critical
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Protein expression

• Expression in mammalian cells
• All large scale therapeutics produced in
  mammalian cells are secreted.
• Scale-up of transient expression Durocher
  Giesse Wurm.
• Recent results 293E cells grow in disposable
  Fernbach flasks to 107/mL, without requiring more
  DNA for transfection.
• The DNA purification method can make a difference
  in transfection (try different kits).
• Testing various commercial media can yield big
  improvements.

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Protein expression

• Expression in yeast cells
• Excellent for secreted proteins, sometimes for
  cytoplasmic proteins.
• The yeast ? mating factor functions for secretion
  and is Gateway compatible for both P. pastoris
  (Esposito) and K. lactis.
• The amino terminus may not be native (often
  leaves EAEA).

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Protein expression
New expression hosts Kluyveromyces lactis (yeast,
galactose induction)
Expression of active interferon (Hy3) in K.
lactis (Zoon)
Interferon in culture medium
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Protein purification

• Our two most common protein formats
• 1. His6- solubility tag - TEV- gene- stop
• Purification procedure
• 1st IMAC ? TEV digest ? 2nd IMAC ? more columns
  as needed.
• Properties
• N-terminal solubility tag (GST also dimerizes
  (e.g., Stevens)
• TEV site can leave native N-terminus (ENLYFQ/X, X
  any amino acid but proline (Waugh)

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Protein purification

• 2. ATG- gene- His6- stop (expression in cell
  culture)
• Purification procedure
• IMAC ? more columns as needed.
• Properties
• Non-removable C-terminal tag.
• Epitope for assessing expression and
  purification.
• Needed a better tag-removal protease for the
  C-terminus X/ABCDEF.

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References
AddGene http//www.addgene.org/pgvec1 Alwine,
NIH seminar Nov. 16, 2005, at http//videocast.nih
.gov/PastEvents.asp, search Bayley, et al., Nat
Chem Biol 2(6), 314-8, 2006 Bayley, et al.,
Biotechniques 25(5), 764-70, 1998 Bayley, et
al., Methods Mol Biol. 182, 139-47,
2002 Beckwith, J Bact 187, 2983-2991
2005 Blattner, et al., Science 312 (5776),
1044-1046, 2006 Butler, Appl Microbio. Biotechnol
68, 283, 2005 Durocher, et al., J Biotechnol.
Epublished Oct 27, 2006 Esposito, et al., Gateway
and Pichia, Protein Expr Purif 40 (2), 424-8,
2005
74
References
Esposito, Cloning pipeline, Meth Molec Biol in
press Giesse et al., Methods Mol Biol. 308, 87,
2005 Graham, et al., FASEB J. 16, 869,
2002 Jarvis, et al., Nature Biotechnology 23,
567575, 2005. Mammalian Gene Collection
http//mgc.nci.nih.gov/ Novagen catalog, cloning
site info for pBACsurf-1 Nierhaus, et al.,
Nucleic Acids Res, 34, No. 19 e135 2006 NCBI
accession number for baculo genome
NC_001623 Promega intron reference p. 6 of pdf
at http//www.promega.com/tbs/tb206/tb206.pdf Ren,
et al., Stem Cells 24, 1338, 2006 Schmidt, Appl
Microbiol Biotechnol 65, 363, 2004
75
References
Stevens, et al., Biochem J 347 Pt 1, 193-7,
2000. Studier, Protein Expr Purif 41(1), 207-34,
2005 Waugh, et al., Biochem Biophys Res Commun,
294(5), 949-55, 2002 Wurm, et al., Methods Mol
Biol 308, 87-98, 2005 Zufferey et al., Journal of
Virology 73, 2886, 1999
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Mammalian expression tips

• Expression in mammalian cells
• For large-scale we use the Corning 3L non-
  baffled Erlenmyer 431252.
• The Corning roller bottle number is 430195.
• The maximum density in the roller bottles is
  2-2.5x10e6/ml and the max. density in the erl. is
  9-10x10e6/ml.
• Maximum volume in the Erl. is 1L and 150-200ml in
  the roller bottle.
• Our 293E line is designated 293 C18 and obtained
  from the ATCC.

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Mammalian expression tips

• The PEI is obtained from Polysciences, linear
  form MW25k. Each batch made (1mg/mL in H2O)
  should be titrated with the DNA. The
  transfections are set with DNA at 1µg/ml. The
  first batch of PEI worked best at a DNAPEI of
  14 and the second batch best at 15 as
  determined by transfection efficiency using
  GFP. Call Butch Hopkins for more details
  (301-846-5713)!
• 1.5 ?g DNA per mL culture.
• Transfection efficiencies usually run around
  50-60 with the 293E line and 5-10 lower with
  the 293T in our hands.

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Mammalian expression tips

• Most serum-free media will totally inhibit
  transfection due to additives, probably
  anti-clumping agents. The Gibco Free-Style
  medium has been specifically designed to not
  inhibit but our cells don't like to grow in this
  medium so the transfection is set in this medium
  and following a four-hour incubation the culture
  is fed with an equal volume of Hyclone HEK293
  medium.
• Add DNA to 1 vol saline per 20 vols medium, then
  PEI, vortex, let stand at RT 15 min, add to cells.