SOLUBLE PROTEIN
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Soluble Protein Expression Optimization PowerPoint PPT Presentation


In many cases, expression of recombinant proteins often results in insoluble and/or nonfunctional proteins. Here, factors in soluble protein expression optimization and several strategies to improve the solubility of the expressed protein are reviewed.

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Soluble Protein Expression Optimization

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Soluble protein expression optimization

SOLUBLE PROTEIN

SOLUBLE PROTEIN

EXPRESSION OPTIMIZATION

EXPRESSION OPTIMIZATION

BiologicsCorp

BiologicsCorp

www.biologicscorp.com

www.biologicscorp.com

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Soluble protein expression optimization

OUTLINE

OUTLINE

Factors in soluble protein expression

optimization

 Codon optimization

 Strains

 Vectors

 Protein synthesis rate

 Growth medium

 Fusion tags

 Co-expression proteins

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Soluble protein expression optimization

CODON OPTIMIZATION

CODON OPTIMIZATION

 Adjust codon usage frequency and enhance mRNA stability

 State-of-art algorithms

 Pre-computed software from a predicted group of highly

expressed genes

Parameters

• Avoided restriction enzymes

• Non-coding regions

• Motifs

• Codon usage bias

• GC content

• mRNA secondary structure

• Repeat sequence

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Soluble protein expression optimization

STRAINS

STRAINS

BL21 (DE3)

BL21 Star (DE3)

Rosetta (DE3)

• Routine protein

expression

• Deficient in proteases

Lon and OmpT, which

increase protein

stability

• Promotes high mRNA

stability and protein

yield

• T7 promoter-based

expression systems

• Contain rare codons

(AUA,AGG,AGA,CU

A,CC,GGA) in E. coli

• Adjust codon usage

bias and enhance

protein expression

BL21 (DE3) pLysS

OrigamiB (DE3)

BL21-CodonPlus (DE3)

• Contain extra copies

of tRNA genes

• Improve expression of

heterologous proteins

from organisms without

AT- or GC-rich genomes

• Combine desirable

characteristics of BL21,

Tuner™, and Origami

strains in one strain

• Ideal for expressing

protein with disulfide

bonds

• Suppress basal

expression

• Ideal for toxic protein

expression

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Soluble protein expression optimization

VECTORS

VECTORS

pET vector

• Powerful: high expression levels, tight control over basal expression

• Versatile: choices of tags and multiple cloning sites

• Rapid: convenient restriction sites for sub cloning, one-step purification

pCold vector

pCold vector

• Efficient: improved protein expression at low temperatures

In-house fusion vector

• Multiple vectors available to improve protein expression level

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Soluble protein expression optimization

PROTEIN SYNTHESIS RATE

PROTEIN SYNTHESIS RATE

 Multiple strategies slows down translation process for

optimized protein solubility.

Use a weak promoter

Use a low copy number plasmid

Lower growth temperature

Lower inducer concentration

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Soluble protein expression optimization

GROWTH MEDIUM

GROWTH MEDIUM

 Special chemicals in media serve play important roles in

protein proper folding and protein stability.

Add buffer

Control pH fluctuation in the growth medium

Add Glucose

Repress induction of the lac promoter by lactose

Add polyols

Work as osmo-prectants and stabilize protein structure

Add prosthetic

groups or co-

factors

Trace metals, minerals, and vitamins may contribute to

protein proper folding and stability.

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Soluble protein expression optimization

FUSION TAGS

FUSION TAGS

 A large variety of protein tags are available for improving

protein stability and facilitating soluble protein expression.

Tag

Size

Protein

Key features

GST

211 aa

Glutathione-S-transferase

Easy to manipulate

MBP

396 aa

Maltose-binding protein

Available for purification

NusA

495 aa

N-utilization substance

Increase membrane protein solubility

Trx

109 aa

Thioredoxin

Simple initial purification step

SUMO ~100 aa Small ubiquitin modifier

High specific cleavage sites

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Soluble protein expression optimization

CO

CO- -EXPRESSION PROTEINS

EXPRESSION PROTEINS

 Chaperones: transiently interact with protein folding

intermediates and contribute to protein expression

 GroES-GroEL

 DnaK-DnaJ-GrpE

 ClpB

 Trigger factor

 Heat shock proteins

 Foldases: accelerate rate-limiting steps in the folding process

 Peptidylprolylcis/trans isomerases (PPI's)

 Disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC)

 Protein disulfide isomerase (PDI)

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Soluble protein expression optimization

PROJECT PROCESS

PROJECT PROCESS

 Soluble protein expression

optimization process

 Codon optimized gene synthesis

 Expression condition

optimization

 Scale-up production

 Purification and delivery

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Soluble protein expression optimization

CONTACT US

CONTACT US

 BiologicsCorp (BIC) owns a self contained and complete optimization platform for

supernatant protein expression. We also offer tag-free protein production, endotoxin

removal, and high-throughput protein production services. Please feel free to contact us

for more information about the services.

Email: [email protected]

  Online Form

Tel: +1 (317) 703-0614

Fax: +1 (855) 427-1516

REFERENCES

REFERENCES

Berrow, Nick S., et al. "Recombinant protein expression and solubility screening in

Escherichia coli: a comparative study." Acta Crystallographica 62.10(2006):1218–1226.

Redwan, El Rashdy M. "The optimal gene sequence for optimal protein expression in

Escherichia coli: principle requirements." Arab J Biotechnol 9(2006):493-510.

Malhotra, A. "Tagging for protein expression. " Methods in Enzymology 463(2009):239-58.

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