Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli

Devendra H Shah; Kenneth D Cain; Gregory D Wiens; Douglas R Call

doi:10.1007/s10126-008-9111-z

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Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli

Journal article

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Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli

Devendra H Shah, Kenneth D Cain, Gregory D Wiens and Douglas R Call

Marine biotechnology (New York, N.Y.), Vol.10(6), pp.719-730

11/2008

DOI: https://doi.org/10.1007/s10126-008-9111-z

Handle:

https://hdl.handle.net/2376/104755

PMID: 18551344

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Abstract

Recombinant Proteins - metabolism

Codon - metabolism

Protein Engineering - methods

Hemolysin Proteins - genetics

Bacterial Proteins - genetics

Flavobacterium - genetics

Solubility

Vibrio parahaemolyticus - metabolism

Recombinant Proteins - genetics

Models, Statistical

Vibrio parahaemolyticus - genetics

Escherichia coli - genetics

Cloning, Molecular

Escherichia coli - metabolism

Bacterial Proteins - metabolism

Gene Expression Regulation, Bacterial

Inclusion Bodies - metabolism

Hemolysin Proteins - metabolism

Flavobacterium - metabolism

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.

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Title: Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli
Creators: Devendra H Shah - Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA
Kenneth D Cain
Gregory D Wiens
Douglas R Call
Publication Details: Marine biotechnology (New York, N.Y.), Vol.10(6), pp.719-730
Academic Unit: Center for Reproductive Biology; Veterinary Microbiology and Pathology, Department of; Paul G. Allen School for Global Animal Health
Publisher: United States
Identifiers: 99900546946001842
Language: English
Resource Type: Journal article