Heterologous protein production in Escherichia coli:strategies and challenges

François Baneyx

Department of Chemical Engineering and BioengineeringUniversity of Washington, Seattle WA

Bottlenecks to efficient protein expression in E. coli

Promoter choice and design

Inefficient transcription No or little protein synthesized

Codon usageTranscript stabilityTranscript secondary structure

Improper secondary, tertiary or quaternary structure formationInefficient or improper disulfide bridge formationInefficient isomerization of peptidyl-prolyl bonds

Inefficient translation No or little protein synthesized

Inefficient folding (cytoplasmic or periplasmic)

Inefficient membrane insertion/translocation

Toxicity Cell death

Aggregation or degradation

Aggregation or degradation

Escherichia coli

Intracellular environment and protein synthesis

Folding modulators of E. coli

Molecular chaperones are a class of proteins that help other polypeptides fold or reach a proper cellular location without becoming part of the final structure

Many - but not all - cytoplasmic chaperones are heat shock proteins transcribed at high level by E32

The 32 regulon consists of ≈ 30 heat shock proteases and chaperones:

DnaK-DnaJ-GrpE (Hsp70/40 family)GroEL-GroES (Hsp60/10 family)ClpB (Hsp100 family)HtpG (Hsp90 family)IbpA-IbpB (sHsp family)Hsp33Hsp31

Foldases are a class of proteins that accelerate rate-limiting steps along thefolding pathway

Thiol/disulfide oxidoreductases catalyze disulfide formation and isomerizationPeptidyl-prolyl cis/trans isomerases catalyze the trans to cis isomerization of X-Pro bonds

“Folding” chaperones

“Disaggregating” chaperone

“Holding” chaperones

?

Folding chaperones in de novo folding

Aggregate

3' 5'

K

TF

J

Native

K

ADP

GrpE

J

GroEL

GroES

ATP

ATP

ADP

ATP

ADPGrpE

DnaK-DnaJ co-expression and low temperatures improve preS2-S’--galactosidase folding

30ºC 37ºC 42ºC

tac preS2 lacZS’

GroEL-GroES co-expression and low temperatures improve leptin folding

However, this strategy does not always work

3' 5'

K

TF

J

Native

K

ADP

GrpE

J

GroEL

GroES

ATP

ATP

ADP

ATP

ADP

GrpE

IbpA/B

Hsp33

Hsp31

ClpBDisaggregation

Holding

Chaperone-assisted protein folding

E. coli Hsp31 mechanism of action

T decreaseT increase

To His-tag or not to His-tag...

Transfer of growing cells from 37 to 10-15oC triggers the cold shock response

Cells growth and protein synthesis stop and resume at lower rates after 1-4h

A subset of cold shock proteins (CspA, CspB, CspG, CspI, CsdA, RbfA) is induced over 10-fold

A subset of proteins involved in housekeeping transcriptional/translational control (IF-2, NusA, HN-S, Pnp, GyrA, RecA) is induced 2-10-fold

The cold-shock response and CspA

Use of rbfA mutants abolishes cspA promoter repression

cspA-driven transcription allows the production of a toxic and proteolytically-sensitive protein in full-length form

cspA-driven transcription allows the production of a poorly translated protein in a partially soluble form

pMM101 + pTG10 pMM101 + pDnaK/J pMM101 + pGroESL

A combination of cspA-driven transcription and DnaK/J co-expression transiently increases IL21 solubility

Making disulfide bridges in the E. coli cytoplasm

Stable disulfide bonds form in the cytoplasm of surprisingly healthy trxB and

trxB gor (sup) strains

Incubation of trxB cells at low temperatures greatly increases oxidation

efficiency

Certain active site mutants of thioredoxin 1 (trxA) are able complement a null mutation in yeast PDI

Purified thioredoxin exhibits PDI activity in vitro

ColE1-compatible plasmids for cspA-driven synthesis of thioredoxin 1 active site mutants

37ºC to A600 ≈ 0.4

IPTG

1h at 37ºC 37ºC

15ºC

IAA IAAIAA

Wild type trxB trxB gor

Effect of mutant thioredoxin co-expression on the recovery of active MalG17-PhoA in wt, trxB and trxB gor cells

Low temperature co-expression of thioredoxin 1 CGHC mutant in an oxidizing background enhances IL21

solubility and stability

Acknowledgments

Dr. Jeff Thomas Paulene QuigleyDr. Jess Vasina Wim HolMirna Mujacic Dr. Kerri Cooper Joanne PalumboStephanie RichardsonDr. Konstantin KorotkovYan BrodskyDr. M.S.R. Sastry

National Science FoundationAmerican Cancer Society

ZymoGenetics