How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

Abstract

Background: In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443-448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/ beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies.

Polyclonal Antibodies to the Coat Protein of Carnation etched ring virus Expressed in Bacterial System: Production and Use in Immunodiagnosis†

“…The best expression level was observed when 0.5 OD culture of transformed BL 21 cells was incubated with 1 mm IPTG at 28°C for 3 h. These conditions favoured the most of the expressed protein to be present in soluble fraction in the cell lysate, while at 37°C most of the expressed protein was present as insoluble fraction. Koschorreck et al (2005) have also reported that when (Thomas and Baneyx, 1996;Bragard et al, 2000;Kadkhodayan et al, 2000;Saini and Vrati, 2003), 0.4 mm (Jacob and Usha, 2002) for expression of Cardamom mosaic virus CP and 0.1 mm (Petrzik et al, 2001) for expression of PNRSV CP in E. coli. While in our case, 1 mm IPTG concentration gave the best results.…”

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies

“…Aggregation is difficult to prevent and even to predict on the basis of the amino acid sequence or other protein features, although a few systematic studies have appeared in recent years [9]. Thus, strategies for controlling protein solubility strongly depend on the specific protein and have to be developed case by case with a time-consuming trial and error approach [1,10].…”

Comprehensive bioinformatics analysis of cell‐free protein synthesis: identification of multiple protein properties that correlate with successful expression

“…In a recent study, E. coli cell-free extract was used to screen for well-expressed and highly soluble candidates from a large number of recombinant proteins (11). Recently, computational approaches to predict protein propensity for expression and solubility in an E. coli host have been reported (2,(12)(13)(14)(15)(16). Depending on the expression conditions and nature of the proteins analyzed in these studies, various physicochemical features were implicated as determining factors for an open reading frame (ORF) to yield a soluble expression product in bacteria.…”