Expression of recombinant peptides in E.coli. Richard Williamson Department of Biosciences University of Kent

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Expression of recombinant

peptides in

E.coli

Richard Williamson

Department of Biosciences

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Introduction – different expression strategies available

Insoluble fusions with KSI

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Why recombinant expression?

Cost Synthesis expensive for large amounts of peptide >30 residues

Fidelity Post-synthesis purification increasingly difficult for peptides over 30 residues

Isotopic Labelling

Enables more sophisticated experiments. 15N and/or 13C often

needed to make unambiguous assignments in unstructured peptides.

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1. Direct Expression

(individual and tandem constructs)

Eg: Human proinsulin (Shen 1984, PNAS, 81, p4627)

Poor stability

Stability much better with tandem repeats (3 or more) where product formed insoluble inclusion bodies

Low expression.

Soluble peptides would be difficult to purify.

Strategies used for the recombinant expression of peptides

in

E.coli.

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2. Soluble Fusion Partner

Fusion partner highly expressed

Improved peptide stability and solubility

Fusion partner can provide a method of affinity purification.

Yield dependent on MW of fusion partner:

Highest expression for GB1 (15-50mg/l purified peptide). Low yields for GST or MBP (<2mg/l peptide)

Peptide cleavage usually enzymatic (factor Xa and SUMO protease give authentic N-terminus)

GB1 has been used as an attached “solubility enhancement tag” for proteins and peptides. Zhou et al 2001, J Biomol NMR 20, p11

Maltose binding protein (MBP) 42kD NEB

Glutathione S-transferase (GST) 27kD Amersham

Thioredoxin (Trx) 12kD Novagen

Small ubiquitin-like modifier (SUMO) 11kD Invitrogen

B1 domain of protein G (GB1) 6kD Koenig et al 2003, J

Biomol NMR, 26 p193

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3. Insoluble Fusion Partners

(Inclusion bodies)

Further improvement in stability and expression yields

Production of toxic peptides

Simple protein isolation

Addition of His-tag for affinity purification in denaturing conditions.

Peptide cleavage usually chemical (CNBr, dilute acid for Asp-Pro)

Only KSI vector commercially available

Expression yields typically >100mg/l of fusion protein. Peptides yields of 5-58 mg/l reported.

Ketosteroid Isomerase (KSI) 14kD Novagen

Oligonucleotide binding domain (SFC120) 14kD Osbourne et al 2003 Anthanilate synthase 36kD Jones et al 2000 RNA binding domain of hnRNCP1 13kD Sharon et al 2002

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Expression of Integrin binding peptides

RGD-containing peptides originally derived from the foot and mouth disease virus capsid coat protein and the latency associated peptide of TGFβ1 and 3

1

10

A20fmvdv2

NAVPNL

RGD

L

QV

L

AQKVART

A20lap2

GFTTGR

RGD

L

AT

I

HGLNRPF

TGFβ3

NEDDHQ

RGD

L

GR

L

KKQKDHH

DBD-1

EK

C

PNL

RGD

L

QV

L

AQKV

C

RT

DBD-2

C

YVPNL

RGD

L

QV

L

AQKVAK

C

Binding motif

RGD

L

xx

L

Insoluble KSI fusion system chosen for recombinant production.

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Expression Strategy (pET31b)

T7lac KSI His6 AlwNI pET31b KSI His6 ATG TAC M TAC ATG M AlwN1 cleavage Annealed synthetic

oligos Ligation TAC

ATG

TAC ATGTAC ATG

KSI ATG M His6 TAC M TAC ATG TAC ATG TAC ATG KSI His6 ATG TAC M M TAC ATG Ligation of cut vector with oligos to give single or tandem inserts.

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)

n Tandem repeats M M P P P P

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Protein expression M M M M H6 KSI Insoluble inclusion bodies

Inclusion body isolation and protein purification. Cyanogen bromide (CNBr) cleavage.

KSI

+

Pep Pep Pep

Pep

3x

+

H6

Lyophilisation and peptide extraction from insoluble KSI using aqueous buffer.

Reverse-phase HPLC.

Pep

Cleavage at Met residues

Pure peptide with homoserine (HS)

modification at C-terminus

HS

HS HS

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Chemical Cleavage Site

The specificity of the AlwNI restriction enzyme allows the vector to be engineered with different residues for chemical cleavage.

…CAG

NNN

CTG…

…GTC

NNN

GAC…

AlwNI

Methionine (ATG) widely used as cleavage with CNBr is highly efficient. Cysteine or tryptophan are alternatives if the peptide contains Met.

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Tandem Repeat Insertions

Tandem inserts for small peptides (<25 residues) increase yield.

Optimal number of inserts = 2-4 (Hartmann et al 2008, J. Biotech 135, p85; Sharpe et al 2005 Prot Expr. Purif. 42, p200)

Annealed oligos can be ligated

together and the desired concatamer gel purified.

Alternatively, tandem inserts (up to 3) can be found be screening

transformations of 10:1 insert/vector ligations using annealed oligos.

500 400 300 200 100 1 2 3 4 5 Mks bps Ligated insert (63 bps) 1 2 No. tandem repeats

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Expression and Purification

66 45 36 29 24 20 14

Mks total lysate pellet supnt washed IBs

Insoluble expression

Cell lysis followed by inclusion body washing gives recombinant product >80% pure.

CNBr cleavage directly on washed Inclusion bodies kDa KSI-peptide (x2) fusion protein 1 2 3 IBs wash

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Cyanogen Bromide Cleavage

66 45 36 29 24 20 14 x1 x1 x2 x3 x3 Number of inserts x3 x2 x1 KSI

Cleavage in 80% formic acid

(inclusion bodies soluble under these conditions)

Cleavage highly efficient.

Reaction diluted and then lyophilised

kDa

Expected position of fusion protein

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Extraction from lyophilised CNBr digrest

Hydrophilic peptides extracted with aqueous buffers (16h, stirring)

Hydrophobic peptides extracted with acetonitrile/water/TFA mixtures.

Peptide further purified by preparative rpHPLC

His

Tag

Peptide-HS Peptide-HSL

Reverse-phase HPLC of extracted 20-mer peptide using phosphate buffer

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Conversion / Modification

Peptides generated with a mixture of homoserine and homoserine lactone at their C-terminus. OH CH2 CH2 CH COOH NH O H2C CH2 CH C NH O Homoserine Homoserine Lactone

If modified residue at the C-terminus is undesired – a stop codon can be added before the His-tag.

Homoserine lactone can be hydrolysed to homoserine by treatment with NaOH

(0.2M for 16h)

Homoserine can be lactonized to homoserine lactone by treatment with TFA (Offord (1972), Biochem J., 129, p499)

Lactonized peptide can be easily modified by amine-containing substituents. Derivatives of biotin, fluorescein, ANS, etc, are available from Molecular probes.

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Yields

Peptide A20fmdv2 A20lap2 DBD1 TGFβ3 No. tandem Repeats 1 2 3 2 2 2 Yield of fusion protein (mg/l) 160 180 (130) 240 80 (70) 80 (65) (70) Yield of purified peptide (mg/l) 16 19 (7) 24 30 (15) 7.4 (5.3) (10)

Expression yields from LB medium shown in black; minimal medium in blue

Yields comparable to other workers using KSI-fusions

Modest increase in yield with tandem inserts.

Losses predominantly at the extraction stage after CNBr cleavage (typically 30-50% efficient).

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Heteronuclear Saturation Transfer Difference (STD) NMR

Integrin (αvβ6)

RGD peptide ligand

Valuable when there is no structural information about the target protein or complex.

STD Intensity

Peptide Sequence

xRGD

L

XX

L

xxxx

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A problem with 1D STD NMR for peptide ligands is signal overlap. Data is acquired from the unbound peptide that is often in a random coil conformation.

ST Reference STD with EDTA ST Difference

6L,10L,13L

DiCara et al. J. Biol. Chem. 2007, 282, 9657-9665.

3V,17V 7R,19R

STD NMR experiment for A20fmdv2 binding to αvβ6

Not possible to distinguish L, V and R shifts

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A solution is to separate into a second dimension… 13C 1H Saturation transfer 1H / ppm 13C 1H Saturation transfer Correlation 1H / ppm 13 C / ppm 2D STD NMR 1D STD NMR

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A solution is to separate into a second dimension… 13C 1H Saturation transfer 1H / ppm 13C 1H Saturation transfer Correlation 1H / ppm 13 C / ppm 2D STD NMR 1D STD NMR

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13C HSQC [0.5h] 13C HSQC with STD editing [43h]

1H / ppm 13 C / ppm

2D heteronuclear-edited Saturation Transfer Difference (STD) NMR (13C)

αvβ6 integrin (10 µM) + 13C-labelled A20fmdv2 (2 mM) 18A 17V 16K 20T 6L,10L, 13L 1H / ppm

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Also 15N-edited two-dimensional STD NMR

avβ6 integrin (10 µM) + 15N-labelled A20fmdv2 (2 mM)

2D 15N edited STD NMR [29h]

Backbone 15NH only.

Nucleus-specific data from 15N and/or 13C-labelled peptides will enable more

precise mapping of the ligand in close contact with the target protein.

Better insight into the binding interaction and conformation of bound peptide.

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Acknowledgements

University of Kent: Jane Wagstaff Mark Howard Kevin Howland Michelle Rowe

CRUK Tumour Biology Centre - Queen Mary’s College

John Marshall

Funding

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Lactoferrin (12 aa) 6mg/l (LB)

Beta-amyloid (16 aa) 10mg/l (MM) Alpha-factor (13 aa) 56mg/l (LB) LF12 (11 aa) 6mg/l (LB)

Figure

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References

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