Abstract
The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2893-y) contains supplementary material, which is available to authorized users.
Highlights
The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing associated with non-secreted proteins (Mergulhao et al 2005)
We show that the type of signal peptide can affect both the structure of the N-terminus and the expression level of recombinant proteins that are secreted from an E. coli expression system
Cloning and secretion of recombinant Csn containing two different signal peptides on agar plates The tac promoter was used to control the expression of the two recombinant Csn forms, and protein over-expression was induced with IPTG
Summary
The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing associated with non-secreted proteins (Mergulhao et al 2005). The aim of this study was to investigate the effect of two different signal peptides on recombinant protein expression and N-terminal processing using chitosanase (Csn) from Bacillus subtilis as a model protein This enzyme is biotechnologically important because it converts chitosan, Pechsrichuang et al SpringerPlus (2016) 5:1200 a recalcitrant waste product from the seafood industry, into value-added chito-oligosaccharides (CHOS), which have been shown to have excellent health and agricultural benefits (Pechsrichuang et al 2013; Zhou et al 2015). It is a relatively small, 28-kDa extracellular enzyme that is secreted from Bacillus subtilis, a Grampositive bacteria (Pechsrichuang et al 2013). Protein expression levels and enzymatic activity at different time points, in various compartments, and the sequence of the N-terminus of the secreted proteins were determined
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