Proteolytic Sites at Protein Termini of BT Cytolytic Cyt2Aa2 Protein Affect its Expression in Escherichia coli
TL;DR
Although the amino acid residues at N- and C-terminal regions of Cyt2Aa2 are eliminated from active protein, they are necessary for protein production and for preventing toxicity against E. coli during heterologous expression.
Abstract
Bacillus thuringiensis (Bt) is well-known for its bio-larvicidal properties. Cytolytic (Cyt) protein is one of Bt larvicidal proteins. This protein requires proteolytic activation to remove partial N- and C-terminal regions. In this study, to improve proteolytic cleavage specificity, proteinase K cleavage sites at both end termini of Cyt2Aa2 protein were substituted by a trypsin cleavage sites L33R, S37K, S228R, and F237K. Afterward, the engineered Cyt2Aa2 proteins were heterologous expressed in Escherichia coli. Most of the mutants were produced as inclusion proteins similar to the wild type but their capability of solubilization and trypsin activation was significantly reduced, particularly for L33R, S37K, and F237K. The S228R mutant could be partially solubilized and activated. Moreover, the new trypsin cleavage site at N-terminus of L33R resulted in an aberrant toxic Cyt2Aa2 protein against E. coli. The L33R mutant limited E. coli growth during protein synthesis. Remarkably, although the capability of solubilization of the mutant proteins was reduced, their mosquito larvicidal activity (except L33R and L33R/F237K) was comparable to the wild type. These findings demonstrate that although the amino acid residues at N- and C-terminal regions of Cyt2Aa2 are eliminated from active protein, they are necessary for protein production and for preventing toxicity against E. coli during heterologous expression.