Overproduction of Recombinant Proteins in Bacteria: Recent Advances in Expression, Refolding, Purifi

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Overproduction of Recombinant Proteins in
BacteriaRecent Advances in Expression,
Refolding, Purification, and TagsRichard
BurgessUniversity of Wisconsin-Madisonburgess_at_on
cology.wisc.eduColorado State UniversityJune
12, 2009
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My Background1. PhD in Biochemistry and
Molecular Biology, 1969 2. James D. Watson
Professor of Oncology in McArdle Laboratory for
Cancer Research 3. Research focused on RNA
polymerase and transcription factors, their
purification, characterization and role in
regulation of gene expression4. Founded
directed UW Biotechnology Center (1984-96)5.
Instructor, Onc. 675 - Protein Purification
(1986-present)6. Editor-In-Chief of journal,
Protein Expression and Purification
(1994-present)7. Instructor and Chair, Cold
Spring Harbor Course on Protein Purification and
Characterization (1992-present)8. Editor of
Guide to Protein Purification, 2nd Ed Methods
in Enzymology, in prep. 2009 (with Dr. M.
Deutscher)9. I love to purify proteins!
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Overexpression of Recombinant ProteinsE. coli
and pET system
Old approach - start with large amounts of
naturally occurring material New approach - clone
the gene, make expression strain, overproduce to
levels of 10-40 of total protein, purify Many
expression hosts - E. coli, yeast, insect cells,
mammalian cells Many expression vectors - pET
most widely used (Bill Studier) Many recent
advances to get high level expression Often get
gt100 mg of protein from 1 liter of culture (3 gm
wet cells) Overproduced protein often in
insoluble inclusion body - can refold Purification
tags - fused to cloned gene to aid in
purification, detection
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pET Expression System
Developed by Bill Studier (Meth Enzymol 185 60,
1990), available thru Novagen Host
BL21(DE3)pLysS - E. coli B, lacks Lon and OmpT
proteases, contains l lysogen (DE3) carrying T7
RNA polymerase gene under lacUV5 promoter
control, contains pACYC-based plasmid (CmR),
pLysS, that makes T7 lysozyme at low
level Vector e.g., pET11a - pBR322-based
plasmid (AmpR), contains T7 promoter with
downstream lac operator, contains LacIQ gene to
make lac repressor (I), contains the epsilon
translation enhancer. When Uninduced Gene
expression is kept very low by lacO just after
lacUV5 promoter (driving expression of T7 RNAP
from lysogen) and the T7lacO promoter on vector
(driving target gene). In addition, residual T7
RNAP is inhibited by low level expression of T7
lysozyme When Induced with IPTG Repression
abolished, T7 RNAP level overcomes lysozyme
inhibition, get efficient transcript and
translation from the plasmid-carried target gene
with copy number of 20-50
T7 RNAP
IPTG
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Recent Advances in E. coli Overproduction
1. Rare codons prevent high level expression
Stratagene strain BL21CodonPlus RIPL
supplements rare Arg, Ileu, Pro and Leu tRNAs.
(Also Novagens Rosetta strain) 2. Some genes
have mRNA that is rapidly degraded in E. coli,
protein level low Invitrogen strain BL21
Star has defective RNaseE that prevents mRNA
degradation and increases protein expression
levels 3. Cant get good overproduction of
disulfide-containing proteins Novagen
Origami strain causes a more oxidizing cytoplasm,
allow disulfide bond formation, can also target
gene product for secretion to periplasm using the
pelB signal sequence 4. Gene not completely
repressed, cant clone very toxic proteins
Epicentre has a CopyControl plasmid developed by
W. Szybalski keeps vector low copy number, can
be induced to high copy number. Novagen has
new medium, OvernightExpress (Studier) that
keeps gene off until glucose used up, then
automatically induces to high levels.
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Article Stratagene Newsletter, Strategies,
16112, 2003. www.stratagene.com
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Article Stratagene Newsletter, Strategies,
16112, 2003. www.stratagene.com
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Unattended high-density cell growth and induction
of protein expression with the Overnight
ExpressTM Autoinduction System (Grabski et al,
Novagen Newsletter, Innovations, 6/03)
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• Recent References on Auto-Induction
• Studier, Protein production by auto-induction in
  high-density
• shaking cultures, Prot. Express. Purific. 41,
  207-234, 2005.
• 2. Sreenath, Fox et al., Protocols for
  production of selenomethionine
• labeled proteins in 2-L polyethylene
  terephthalate bottles using
• auto-induction medium, Prot. Express. Purific.
  40, 256, 2005.
• Tyler, Fox et al., Auto-induction medium for the
  production of
• U-15N- and U-13C. U-15N-labeled proteins for
  NMR screening and
• structure determination, Prot. Express. Purific.
  40, 268, 2005.

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Recent Advances in E. coli Overproduction
5. Cant get high levels of overproduction of
membrane proteins Avidis has strain (C43)
that overproduces internal membranes, provides
place for membrane proteins to be inserted
(Ref Miroux and Walker, J Mol Biol 260 289,
1996) 6. Expensive to grow many cultures of
cells for proteomics applications Can grow
cells to high density in plastics 2-liter soda
bottle with very rich medium. Gives very good
aeration. Not uncommon to get 10 gm wet weight
cells from 500 ml culture in 2-liter bottle.
(Ref Millard et al, Protein Express. Purific.
29 311, 2003) 7. Need to co-express two or
more proteins of a multi-subunit complex
Several systems that allow co-expression of
several different genes, often leads to assembly
of soluble complex in vivo while expression of
each single gene leads to formation of inclusion
bodies.
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(Novagen Newsletter)
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Other Expression Systems Secretion
E. coli, Bacillus subtilis, Filamentous
fungi (Aspergillus) Yeast
Saccharomyces cerevisiae, Pichia pastoris Insect
cells using baculovirus vector Sf9, Manduca
larvae Mammalian cells CHO cells, HEK293T
cells, NIH 3T3 cells, Transgenic goats,
transformed cow udder Plants Into seeds
(soybeans), transgenic alfalfa, enzyme
farming In vitro translation (Roche,
Promega) Commercial sources of vectors/host
systems Promega, Epicentre, EMD/Novagen, GE
Healthcare, NEB, Invitrogen, Stratagene,
Clontech, Qiagen, Roche
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Expression in Mammalian Cells F.M. Wurm,
Production of recombinant protein therapeutics in
cultivated mammalian cells, Nature Biotech. 22
1393, 2004 S.C. Makrides, Components of vectors
for gene transfer and expression in mammalian
cells, Protein Expression. Purific. 17 183-202,
1999. CHO (Chinese hamster ovary cell
line) First used 1986 - Used by pharmaceutical
industry to produce proteins that require
post-translational modification to express
biological function Often used to try to get
human glycosylation patterns. BUT - pattern can
be dramatically affected by pH of the culture
medium!
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PURIFICATION OF OVER-PRODUCED INSOLUBLE PROTEINS

• Readings
• Burgess and Knuth, Strategies for Protein
  Purification and Characterization CSH Lab Manual,
  pp204-274, 1996
• Mechanisms of Protein Folding, Ed R.H. Pain, IRL
  Press, 1994 Thatcher Hitchcock, Protein
  Folding in Biotechnology, pp 229-61 B. Nall,
  Proline isomerization as a rate limiting step, pp
  80-103 H.F. Gilbert, The formation of
  native disulfide bonds, pp 104-136
• P. Wingfield, Current Protocols Protein Science,
  Unit 6.5, pp 1-27, 1997

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Refolding Proteins from Inclusion Bodies
Inclusion Bodies (IBs) (granules, refractile
bodies) About 70-80 of overproduced proteins are
mostly insoluble Seem to be caused by aggregation
of partially folded (sticky) protein in vivo
under conditions when protein production is too
fast or too high Problem or advantage? Disadvantag
es You have to solubilize and refold.
Often large volumes required during refolding.
Unless refolding is optimized, you
often get low recovery of active, monomeric
protein. (Protein can be soluble, but
multimeric) Concern that refolded protein is not
really native. Advantages rapid and simple
removal of most contaminating material just by
washing IB sometimes prevents proteolysis
The problem is no longer purification but
refolding!
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Strategies to Increase Proportion of Soluble
Protein
Induce over-production at lower temperatures,
e.g. 20oC Add sugars to 0.4 M (Bowden, Biotech
Progress 4 97, 1988) Co-overproduce chaperones
(DnaK, DnaJ, GroEL, GroES) Subject cells to brief
heat shock at 42oC, then shift to 20oC,
induce Co-expression of several subunits of a
complex Fuse to easily refolded protein
Solubility Tags. e.g., NusA, Trx,
MBP, Sumo, HaloTag
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The Challenge of Refolding is to Minimize
Aggregation
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Refolding Proteins from Inclusion Bodies
Typical
Procedure 1. Grow up and induce cells 2. Harvest,
weigh the cell pellet, store frozen 3. Break
cells by sonication (pLysS cells easy to break
due to T7 lysozyme) 4. Spin, wash IB pellet with
1 TritonX-100 to solubilize membranes and
membrane proteins, then wash with buffer to
remove TritonX-100 5. Solubilize IB with
denaturant such as 6 M GuHCl or 0.3 Sarkosyl to
about 1 mg/ml. 8M urea can be used, but can lead
to carbamylation 6. Drip dilute slowly into
15-60 volumes of suitable refolding buffer 7.
Pass dilute refolded protein over a suitable ion
exchange column 8. Wash IEC column and then elute
with a salt gradient.
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• A final IEC step accomplishes five important
  jobs
• It concentrates protein (from 500 ml to 5 ml)
  
• It removes denaturant (in the flow through)
• It removes minor impurities (in flow
  through or binding weaker or stronger to column)
  
• It selects for a homogeneous, active monomer
• It removes soluble protein multimers (they
  tend to elute later due to their higher charge)
• The resulting protein is a homogeneous, active
  protein suitable for crystallization

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09CSH Group2 SigmaF, Gel 1 - 4/20/09
kDa 260 160 110 80 60 50 40 30
20 15 10 3.5
??
?F
M A B C
C Du/2 A/2 B/2 C/2 C/2 Du/4
?F
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Base Buffer 25mM Tris, 0.5mM EDTA, pH
7.9 Refolding 120 flash dilution
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s32 refolded from 3 M GuHCl from 8 M urea
Buffer control
Green Laser to visualize light scattering
(turbidity)
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• SigmaF refolded from 8M Urea into TGE
• Column Poros50 HQ, CV 15ml
• Buffer A TGE
• Buffer B TGE1M NaCl
• Gradient 0-60 Buffer B, 120ml
• Flow Rate 5ml/min, 5ml fractions

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Gel 2-sigmaF, refold from 8 M urea, on POROS 50
HQ col
kDa 260 160 110 80 60 50 40
30 20 15 10 3.5
?F
M Du/2 ON FT A7 A8 A9
A10 A11 B10 B8 B6
(Conc by pptn)
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Refolding Additives Glycerol -
10-50 Arginine - 0.5 M Systematic Searching
for Refolding Conditions Hampton Fold-ItTM
Kit (Varies pH, salt, divalent cation,
other additives such as PEG, sucrose,
GuHCl, red/ox agents) Novagen/EMD iFOLD2,
iFOLD3 kits
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Proline Isomerization can be Rate-Limiting Step
in Refolding
Trans/Cis 70/30 at equilibrium in denatured
protein
Can catalyze with Protein Proline Isomerase (PPI)
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The Formation of Native Disulfide Bonds
(H.F. Gilbert, in R. Pain, 1994)
Formation disulfide bonds catalyzed by Protein
Disulfide Isomerase (PDI)
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Efficient folding of proteins with multiple
disulfide bonds in the E. coli cytoplasm
(Bessette, Aslund, Beckwith, Georgiou, PNAS 96
13703-8, 1999) The environment in the E. coli
cytoplasm is reducing, preventing most disulfide
bond formation Mutations in the glutathione
reductase (gor gene) and thioredoxin reductase
(trxB gene) enhance formation of disulfide bonds
in the E. coli cytoplasm Strains AD494 (DE3) and
BL21trxB(DE3) are trxB deficient Novagen Origami
strains are trxB and gor deficient, but dont
grow very well
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Typical Conditions for Reoxidizing a Refolded
Protein Can re-form disulfide bonds by Air
oxidation - expose to air, without reducing agent
for several days Redox buffer
reduced/oxidized glutathione 10/1 (3 mM GSH/0.3
mM GSSG) Protein disulfide isomerase
Refolding column (Avidis) contains PPI,
PDI New small molecule (BMC), like PDI
(Woycechowsky, Wittrup, Raines, Chem Biol 6
871-79, 1999)
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Engineering Proteins for Ease of Purification and
Detection Once you have a gene cloned and can
over-express the protein, you can alter protein
to improve the ease of purification or
detection You can fuse a tag to the N-or C-
terminus of your protein You can decide to
remove tag or not Basic strategies Add signal
sequence that causes secretion into culture
medium Add protein that helps the protein stay
soluble Add sequence that aids in
precipitation Add an affinity handle Affinity
Tag Add sequence that aids in detection
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(Ford et al., 1991)
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Common endoproteases used to remove protein
tags Protease Cleavage site Enterokinase
DDDDK? Factor Xa IEGR? Thrombin LVPR?GS PreS
cission LEVLFQ?GP (Human rhinovirus 3C
protease) TEV ENLYFQ?G (Tobacco Etch Virus
protease) TVMV ETVRFQG?S (Tobacco Vein
Moulting Virus Protease) SUMO Protease Recognizes
SUMO tertiary structure (Catalytic core of
Ulp1) and cleaves at the C-terminal end of the
conserved Gly-Gly sequence in SUMO
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(Nilsson et al., 1997)
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Common Affinity Tags Tag Size Affinity
matrix His-tag 6-10 His Immobilized metal
ions Ni, Co, Cu, Zn GST 211
aa Glutathione resin (Glutathione
S-transferase) FLAG-tag 8 aa
(DYKDDDDK) Anti-FLAG mAb
Strep-II tag 8 aa (WSHPQFEK) Strep-Tactin
(modified streptavadin) Protein A
280 aa Immobilized IgG (Staphylococcal Protein
A) MBP 396 aa Cross-linked
amylose (Maltose binding protein)
CBP 26 aa Immobilized
Calmodulin (Calmodulin binding protein)
CBD 51 aa Chitin (Chitin binding
domain)  HaloTag 300
aa Chloroalkane
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Ni2-NTA Purification of His6-tagged
Proteins Bind at pH 7.9, 5 mM imidazole to
prevent weak binding of His or Cys-containing
proteins 300 mM NaCl to prevent ionic binding
can bind in presence of 6 M GuHCl or 8 M urea so
can purify protein denatured and then refold it
(you can also refold it on the column) Wash
20-80 mM imidazole. Can wash with 60
isopropanol to remove endotoxins, residual
detergents, some non-specifically bound
proteins Elute 3 different methods 300-800
mM imidazole (most common way) EDTA to remove
Ni2 from NTA lower pH (less that pH 5.9)
where the imidazole group of Histidine becomes
protonated
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Interaction Between Neighboring His residues
on the His6-tag and Ni-NTA Matrix
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Structure of Imidazole and Histidine
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Ni2-NTA Purification of His6-tagged
Proteins Additional Tidbits Ni-NTA binding of
His6-tagged proteins (E. Hochuli in
1987) Aggregate (e.g., dimer) requires higher
imidazole to elute 10-10 M Ni leaches off
column, can accelerate air oxidation of
DTT Unlike what Qiagen says, you can use
(NH4)2SO4 with Ni columns Sometimes you find
bound DnaK and can wash it off with ATPMg2 One
protein in E. coli binds very tightly (SlyD, very
His-rich, 27 kDa) (Roof et al., Molec.
Microbiol. 25 1031, 1997) Can use Cobalt (Talon
columns) or Zinc in place of Nickel Often (but
not always) proteins can be used without removing
His-tag Thiol reducing agents form metal
sulfides, cause column to turn brown Can avoid
by using TCEP
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Courtesy of Paul Blommel (B. Fox Lab)
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GST Fusions
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Using GST-Tagged Proteins and GSH Affinity
Columns Bind to GSH resin Wash Elute
with 10 mM GSH Comments pGEX system,
Amersham Biosciences Remember, GST is a dimer
of 26 kDa monomers GST from Shistosoma
japonicum, (Smith, Johnson Gene 6731,1988)
GST-PreScission Protease (human rhinovirus 3C
protease)
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(Amersham GST Gene Fusion System Booklet, 1997)
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Epitope Tags for Protein Purification Tag
Size MAb
Elution Source HA 9 aa anti-HA low pH
Sigma FLAG 8-23 aa M1 M2 EDTA/peptide
Sigma T7-tag 11 aa anti-T7 pH 2.2
Novagen S-tag 15 aa S protein pH 2
Novagen Softag1 8-13 aa NT73 Salt/polyol Softag2
10-14 aa 8WG16 Salt/polyol Softag3 6-8
aa IIB8 Salt/polyol NeoClone Softag4 8RB13 Sa
lt/polyol (Adapted from Burgess and Thompson,
2002)
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Common Solubility Tags Tag Size Protein
MBP 396 aa Maltose Binding Protein NusA 495
aa N-Utilization substance Trx 109
aa Thioredoxin SUMO 100 aa Small
Ubiquitin modifier GB1 56 aa IgG domain B1
Streptococcus Protein G SET/SEP lt 20
aa Hydrophilic solubility enhancing
peptide sequences HaloTag 300 aa Mutated
dehalogenase
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HaloTag Technology

Irreversible attachment of chemical
functionalities

• Non-equilibrium process no off rate and high
  on rate allows highly efficient protein capture
  and labeling

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Engineering for Covalent Bond

• Evidence for covalent bond
• Stability under denaturing
• conditions
• Mass Spec analysis

Bond stability
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Structural Optimization HT7
H272F H272N
K175M C176G
P291S A292T
S58T
D78G
A155T
N227D
E257K
Y273L
A172T
A224E
Y87F L88M
L47V
E160K A167V
K195N
T264A
C128F
294E 295I 296S 297G

• Increased Solubility
• Increased Stability
• Faster Binding Kinetics
• For use in mammalian cells, in vitro
  expression, and E. coli

Bound TMR-ligand
22 changes
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Increased Binding Kinetics
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Increased Stability

• HT7 Improved stability at elevated temperature

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HaloTag7 Based Protein Purification Method
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Expression / Solubility in E.coli
23 human proteins known to be difficult to express
MAPK14 41.4 kDa
MAPK8 48.4 kDa PRKCG
52.0 kDa Marker
HT7 GST MBP His HT7
GST MBP His HT7 GST
MBP His Med High
KRX cells auto induction (0.05 Glucose 0.2
Rhamnose)
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HT7 Enhances Protein Solubility in E. coli
HT7 yielded soluble protein more frequently than
His-tag, GST or MBP (3.4, 2 and 1.4 fold more,
respectively).
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