ARS | Publication request: Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli

Submitted to: Marine Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 16, 2008
Publication Date: June 13, 2008
Citation: Shah, D.H., Cain, K.D., Wiens, G.D., Call, D.R. 2008. Challenges associated with heterologous expression of Flavobacterium psychrophilum proteins in Escherichia coli. Marine Biotechnology.

Interpretive Summary: Flavobacterium psychrophilum is the etiologic agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome in salmonid fish. High mortality rates and spinal deformities associated with BCWD are responsible for serious economic losses to public and private aquaculture in many parts of the world. Currently, there is no commercial vaccine available for the control of BCWD and immunization of rainbow trout with whole-cell killed F. psychrophilum preparations have, in general, yielded limited success. With the recent availability of the whole genome sequence of F. psychrophilum CSF 259-93, it is now possible to predict antigens that can be cloned and expressed for vaccine efficacy trials. These experiments depend on generating soluble recombinant protein, and this is typically accomplished using a heterologous expression system such as E. coli. However, we predicted that 88.5% of 96 putative virulence associated genes of F. psychrophilum would form insoluble aggregates in E. coli called ¿inclusion bodies¿, and we have experimentally verified that a subset of these protein are insoluble. In addition to this problem, a comprehensive genome analysis identified substantial differences in codon usage between F. psychrophilum and E. coli that is presumably responsible for premature protein truncation and low expression yield. Consequently, we developed an alternative expression host system, utilizing Vibrio parahemolyticus to express F. psychrophilum proteins. Using this system, full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified in soluble form. Thus, V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy difficulties associated with expression F. psychrophilum proteins in E. coli.

Technical Abstract: A two-parameter statistical model was used to predict the solubility of 96 putative virulence associated genes of Flavobacterium psychrophilum (CSF259-93) upon over expression in E. coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates called 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 due to 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, V. parahemolyticus 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. parahemolyticus 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.