optimizing conditions for recombinant soluble protein ... · optimizing conditions for recombinant...

Optimizing conditions for recombinant soluble

protein production in E.coli

Keshav Vasanthavada, MSc., MS

May 8th, 2014

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Presentation overview

Introduction

Before embarking on a protein expression project

E.coli expression

GenScript’s solution for expression optimization

Factors critical to solubility

Conclusion

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About GenScript

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GenScript

Products & Services Make Research Easy

Gene

Peptide

Protein

Antibody

Discovery

Biology

Cell Line

Introduction

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Historical perspective

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1940 1950 1960 1970 1980 1990 2000 2010 2020

1953 – elucidation of

the helical nature of

DNA

(Franklin & Wilkins)

1941 – One Gene One

Enzyme Hypothesis

(Beadle & Tatum)

Solving the 3-D

structure of DNA

(Watson & Crick)

1957 - Central Dogma

(Francis Crick)

1970 – Discovery of

Restriction Enzymes

(Smith & Nathans)

1973 – Birth of

Recombinant DNA

Technology

(Cohen & Boyer)

1977 - First

recombinant protein

(somatostatin) made

in E. coli

1977 – DNA

sequencing

(Maxam & Gilbert)

1980 – PCR was

developed

(Kary Mullis)

1987 – Refolding of

Inclusion bodies from E. coli

(Buchner, Kelley , Rudolph)

1992 – Baculovirus

Expession Vector System

(Summers, 1983; King &

Posse, 1992)

1993 – Affinity purification

of His- & GST-tagged

proteins

(Hochuli 1988; Jones 1993)

Mammalian cell expression

(Hauser 1988; Page 1991;

Geisse 1996)

Full length synthetic

Proteins?

Protein Amplification

Technology?

Introduction

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• Recombinant protein production (RPP) is essential to

functional characterization and structure determination

• Variety of hosts available for RPP

• Scientists faced with overwhelming choices

• Escherichia coli [E. coli] is “work horse”

• Successful implementation of bacterial protein production project dependent on several factors

• Observations & Recommendations for optimizing recombinant protein production in E. coli

• We describe learnings from our collective experience

• GenScript Production Team has shipped over 3,000 batches of recombinant proteins

Introduction to topic

5 Introduction

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Common questions

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My Protein

My Own

My Precious

But me why needs it?

How is me making it?

Activity


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Host considerations

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Bacteria

E. coli

1. “Work horse”

2. Well established

3. High expression

4. Simple genetics

5. Easy scale up

6. Speed

7. Costs

8. Equipment

Insect Sf9, Sf21, S2, High-5

1. PTMs

2. Soluble proteins

3. High expressers

Mammalian CHO, HEK, COS

1. PTMs

2. Soluble proteins

3. Low expresser

4. Expensive

Yeast S. cerevisiae

P. pastoris

1. PTMs

2. Soluble proteins

3. High expresser

Cell Free In vitro

1. Expensive

2. Not reproducible

3. Scalability issues


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92%

3.8% 2.06% 1.03% 0.29% 0%

20%

40%

60%

80%

100%

E.coli Insect Yeast Mammalian Cell Free

PDB entries reflects dominance of E. coli expression

E. coli is the top choice

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Number of entries in the PDB by expression system as a percentage of total number of chains with an

identifiable expression system, as of April 15, 2014. All values are approximate. Reference - http://www.rcsb.org/pdb/home/home.do

E.coli expression

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Advantages Disadvantages

Simple genetics Lack of PTM

Easy to manipulate Codon usage

Inexpensive to culture Inclusion bodies

Easy to Scale up Low yield of many eukaryotic proteins

Fast expression Poor secretion

High yields High MW proteins difficult

E. coli expression – why & why not?

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Low/No

Expression

Insoluble

Expression

E.coli expression

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What is the problem? what are the solutions?

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E.coli

Refold

Optimize

No Target Engineering

• Temperature

• Strains

• Media

• Buffers

• Chaperones

Engineer Target

• Truncate

• Mutate

• Fusion Partner

Inclusion

Bodies

57%

43%

~24,000 Targets NESG Complete

Soluble

Insoluble

E.coli expression

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Variables Desirables

Goal of a protein production campaign

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Generation of high yields of soluble protein in a cost-effective and expeditious manner is the single most important

determinant of a successful protein production campaign

E.coli expression

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Project initiation

Literature Review

Target Analysis &

Engineering

Host Consideration

Cloning scheme/Gene

synthesis

Small scale expression

optimization

Scale up and Purification

Protein expression campaign workflow

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What has been done already? Homologs, truncations, mutations?

Full-length or fragment? Tag or tag-less?

Which tag? N or C-terminus?

E.coli expression

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Target Protein Expression in E. coli

No

Discard

Re-evaluate

Optimize

[PROTentialTM]

Insoluble

Optimize

[PROTentialTM]

Refold

[FoldArtTM]

Soluble

Scale up & Purify

Archive

Flow chart of a bacterial expression project

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WITHOUT target engineering

• Codon optimization

• Promoters

• Vectors

• Temperature & Culture

• Strains

• Media

• Buffers

• Chaperone co-expression

WITH target engineering

• Truncations

• Mutants

• Fusion partners

Good idea to run small scale expression and test purification

E.coli expression

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Eliminate the guesswork from your Protein production work

What is PROTentialTM?

• PROTentialTM is a new protein expression, evaluation and optimization service

offered by GenScript.

What are the applications of PROTentialTM?

• Evaluate whether your target protein expresses in your chosen system.

• Identify the best expression system for your target protein.

• Identify the construct and conditions that give you the most robust soluble expression

of your target protein.

Evaluate before scale-up protein production, to avoid wasting your time & resources

PROTentialTM - expression evaluation & optimization

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One stop service at GenScript: gene synthesis Subcloning PROTentialTM Scale up protein production


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PROTentialTM Standard Package (starting from $280)

• Available for 3 expression systems: bacterial, insect & mammalian

• Protein expression evaluation & solubility test

PROTentialTM Silver Package (starting from $1440)

• Currently for bacterial expression system only

• Test 8 conditions by combining temperature, media components & inducer concentration.

• Allow to identify the best expression condition with your chosen vector & bacterial strain.

PROTentialTM Gold Package (starting from $4800)

• Currently for bacterial expression system only

• Test 48 conditions, by combining parameters including not only those 3 covered in silver package,

but also promoter, host strain, and fusion partner.

• The most robust and high throughput protein expression & solubility optimization matrix.

Add-on item: 1L bacterial expression with 1 step purification at $600/condition (except for Flag-tagged

protein, the cost is $800/condition).

PROTentialTM portfolio

15 GenScript’s solution for expression optimization

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PROTentialTM standard packages

Expression system Price Timeline Deliverables

Bacterial Starting from $280 1-3 weeks

• SDS & WB result • Expression data report

Baculovirus/ insect

Starting from $400 3-4 weeks

Mammalian Starting from $500 3-4 weeks

All 3 systems Starting from $950 3-4 weeks

Each package represents one gene, one vector and one expression system. These standard

packages offer expression and solubility test but no optimization


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Options for protein tag, growth media & vectors

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Mammalian

Protein Tag

His (default)

Fc

Flag

V5

HA

C-Myc

GST

Insect

Protein Tag

His (default)

Fc

Flag

GST

Strep II

MBP

V5

HA

C-Myc

E. Coli

Protein Tag

His (default)

GST

Trx

MBP

SUMO

Flag

Growth Media

LB (default)

TB

2xYT

Vectors

pET system

pGEX system

pCold system

Gene synthesis customers have listed

options with the standard PROTentialTM

packages.

If a modification or tag is not on this list,

PROTentialTM silver or gold package is

required in order to accommodate such

request.



Other optimization parameters (Gold package only)

Promoters

T5, T7, trc, tac, CSPA, AraBAD, Lac, Lac UV5, Trp, pL, T7-lac, T5-lac, Lac-ara1, Ltet0-1,

PhoA, Psyn, recA, proU, cst-1, tetA, cadA, nar, T3-lac, T4 gene 32, nprM-lac,

lpp, lpp-lac, VHb, or others.

Bacterial strains

BL21, BL21(DE3), BL21*(DE3), BL21(DE3) pLysS (E), BL21-SI, BL21-AI, BL21trxB, BL21

CodonPlus, Origami, Origami B, Origami B pLysS, C41(DE3), C43 (DE3), Lemo21(DE3),

Turner, Tuner pLysS, AE, Rosetta, Rosetta pLysS, Rosetta-gami-pLysS, Shuffle, DH5a,

TOP10, JM109, TG1, Stbl2, XL10-Gold, AD494, HMS174, NovaBlue(DE3), BLR , HB2151,

W3110 or others.

Induction conditions

Chosen based on our expertise; Can accommodate customer’s specific request.

Medium components

Chosen based on our expertise; can accommodate customer’s specific request.

Temperature

6C, 10C, 15C, 16C, 20C, 25C, 30C, 37C, or as specified by customer.


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• Vectors & Promoters

• Induction time

• Chaperones & Foldases

• Growth Media • Inducer

I. Codon Optimization

II. Strain

IV. Fusion Partner

III. Temperature


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SOLUBLE

PROTEIN


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I. OptimumGeneTM codon optimization

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GenScript has optimized over 50,000 sequences in all

major expression systems. The expression level of

target proteins can be significantly improved by our

OptimumGeneTM Codon Optimization Technology.



Case study: codon optimization

Fig. 1: Expression Result of Protein α after Codon Optimization. The expression level of Protein α using GenScript’s OptimumGeneTM Codon Optimization is 3 times more than that of competitor’s.

Fig. 2: Expression Result of Protein β after Codon Optimization. The expression level of Protein β using GenScript’s OptimumGeneTM Codon Optimization is 13 times more than that of competitor’s.


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# Expression Strain Rationale

1 BL21(DE3) Work-horse of recombinant protein expression. Lamba DE3 lysogen. Basal exp high. Non-toxic preferred

2 BL21(DE3) pLysS T7 lysozyme to repress basal level expression

3 BL21(DE3) pLysE T7 lysozyme to repress basal level expression. Higher level of repression

4 BL21 Star (DE3) RNaseE (rne131) mutant. Reduced mRNA degradation. Higher stability of transcripts

5 BL21 Star (DE3) pLysS As above, coupled with higher level of repression

6 BL21 (DE3) Codon Plus RIPL Contain extra copies of amino acids R,I,P,L tRNA genes. Supplements for codon deficiencies and bias

7 Origami™ 2(DE3) pLacI trxB and gor reductase mutants. Enhance disulfide bonds in cytoplasm. Rare tRNA genes also

8 Origami™ 2(DE3) pLysS trxB and gor reductase mutants. Enhance disulfide bonds in cytoplasm. Lower basal repression

9 Origami™ B(DE3) pLacI Enhance disulfide bonds in cytoplasm and control IPTG entry into cells

10 Origami™ B(DE3) pLysS As above, coupled with higher level of repression

11 OverExpress C41(DE3)pLysS Uncharacterized mutation that allows higher production of "difficult" proteins

12 OverExpress C43(DE3) pLysS Uncharacterized mutation that allows higher production of "difficult" proteins

13 BL21-Gold (DE3)pLysS Lon and OmpT protease deficient for greater recombinant protein stability. Higher plasmid stability

14 SHuffle™ T7 Express lysY Enhances disulfide bond formation in cytoplasm, DsbC promotes correction of misoxidized proteins

15 Arctic Express™ Co-expression with chaperonins Cpn60 & Cpn10 for increased yield of soluble proteins

16 Rosetta™ 2(DE3)pLysS tRNA genes for 7 rare amino acids plus reduced basal expression

17 Rosetta-gami™ B(DE3)pLysS Above plus increased disulfide bond formation in cytoplasm plus reduced basal expression

18 Tuner™(DE3)pLacI Lac permease lacY mutant allows uniform entry of IPTG into cells, enhances solubility

19 Tuner™(DE3)pLysS Above function plus reduced basal expression

20 Lemo21(DE3) Tunable expression by varying lysozyme concentration, regulated by addition of Rhamnose

II. Strain

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Case study: Impact of E.coli strains on

soluble expression

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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17 18 19 20 21 22 23 24

M

M

M

0.5mM IPTG induced/15C Overnight, Ni-IMAC purified

Lemo21 (DE3) with varying

concentrations of Rhamnose

30kDa

A

20kDa

B

M

0.5mM IPTG induced/15C Overnight, Ni-IMAC purified

Lemo21 (DE3) with varying

concentrations of Rhamnose

STRAIN MAKES A DIFFERENCE


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III. Temperature: case study

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15C RT 37C

65kDa

WB

Lower temperatures result in more soluble protein

Total

Soluble

TEMPERATURE MAKES A DIFFERENCE


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IV. Fusion partner/affinity tag

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Tag ~MW [kDa] Pros Cons

6His ≤1

Universal, small, works

with native and denaturing

conditions

Usually requires more than

1-step elution. Not entirely

innocuous

CBP 4 High specificity Purity differs greatly

Flag 1 High purity

Introduces Enterokinase

Expensive and elution can

be a problem occasionally

GST 26 Elution easy, functions as

solubilizing tag

Dimerizing nature

Leaches occasionally

MBP 40 Solubility enhancer Need longer contact times,

Relatively large tag

Strep ≤1 High specificity, Mild

elution conditions Need longer contact times

NusA 55 Solubility enhancer Very large tag

Trx 12 Solubility enhancer Does not work well with

larger MW target proteins


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Case study: Importance of fusion partner

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1 2 3 4

30kDa

C

15kDa

D

FUSION PARTNERS MAKE A DIFFERENCE


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Case study: Duration of induction

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1. Protein D [0.1mM IPTG, 15C, 3 hours]

2. Protein D [0.1mM IPTG, 15C, Overnight]

3. Protein D [0.1mM IPTG, RT, 3 hours]

4. Protein D [0.1mM IPTG, RT, Overnight]

0 15 30 45 60 75 90 105 120 135 150 165 180

1 2 3 4 1 2

20kDa

E

Minutes

Detectable recombinant protein expression begins as early as 15-30 minutes

50kDa

F

Longer need not always be better

1. Protein E [0.1mM IPTG, 15C, 3 hours]

2. Protein E [0.1mM IPTG, 15C, Overnight]

60kDa

G

Longer can occasionally be bad


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Cloning Strategy

LIC or recombination-based cloning strategy for high throughput & parallel

construct generation

If possible, create expression vectors with N-terminal fusion/solubility

partners for easy cloning scheme

Expression

Express both N- and C-terminally affinity tagged constructs [especially 6His]

In many cases at least one version will express

If checking for enzyme activity, one version might show higher activity than

the other

Purification

Note that C-terminally affinity tagged constructs allow you to purify full

length proteins

You could try double-tagging approach by using one affinity tag [e.g., GST,

MBP at the N-ter and 6His at the C-ter to solubilize and purify full length

protein at the same time

Factors to consider

28 Conclusion

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No simple, global solution to address insolubility issues in E.coli expression

Codon optimization, strain, induction temperature and duration, fusion partners make a difference

Small scale expression study should be predictive for early triage of soluble constructs

Small scale purification to assess protein behavior and avoid hiccups during scale up

Parallel approaches increase the chance of success

Suggested approach –

• Combinatorial tagging at the N-terminus [6His + solubilizing tag]

• Protease Cleavage site for tag removal after 1st round purification

Expression levels, activity, purity, homogeneity, stability are important factors that must be considered

Summary & takeaways

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Solubilizing Tag 6His ATG Protease

Tag Target

Promoter Protease

Linker

Conclusion

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GenScript’s experience in protein expression & purification

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GenScript has delivered over 3,000 proteins in four expression systems with 92% success rate for all protein projects. GenScript has successfully delivered a variety of difficult proteins, including trans-membrane proteins, co-expression proteins, proteases, kinases, cytokines, antibodies and several other proteins to customers

worldwide.

Conclusion

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Thank you for your participation

We wish you all success in your Research

[email protected]

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