Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli.
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.
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA.
Devendra H
DH
Shah
Kenneth D
KD
Cain
Gregory D
GD
Wiens
Douglas R
DR
Call
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003062:Codon / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005417:Flavobacterium / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D015233:Models, Statistical; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D006460:Hemolysin Proteins; D011994:Recombinant Proteins