OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system

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.

A Robust Fractionation Method for Protein Subcellular Localization Studies in Escherichia Coli

“…To assess the extraction of a recombinant secreted protein from periplasm using PureFrac, the periplasmic PhoA (fused to a TorA-signal peptide) was co-expressed with PDI and Erv1p in WT and Tat-null strains. Such cells were cultivated in varying settings (Table2) thatare commonly used to overexpress proteins in the literature [18,19,25,26,32,33,38] to test whether growth conditions affect cross-contamination propensity.…”

“…compared periplasmic fractions obtained by EDTA/lysozyme and cold osmotic shock and demonstrated cross-contamination in each case [17,24]. Many publications show no due-diligence to fraction purity [18,19,21,25,26]. Some do utilize controls in the form of subcompartment specific host proteins as purity markers but are incomplete [27,28].…”

Secretion of recombinant proteins from E. coli

“…Homologous sequences include the signal sequences from LamB [114], MalE [109,115], OmpA [116][117][118][119], OmpC [120], OmpF [121], OmpT [77,122], and PhoA [115,123,124]. Homologous sequences include the signal sequences from LamB [114], MalE [109,115], OmpA [116][117][118][119], OmpC [120], OmpF [121], OmpT [77,122], and PhoA [115,123,124].…”

“…Several homo-and heterologous signal sequences that target proteins to the Sec machinery have already been successfully used for recombinant protein expression in E. coli. Homologous sequences include the signal sequences from LamB [114], MalE [109,115], OmpA [116][117][118][119], OmpC [120], OmpF [121], OmpT [77,122], and PhoA [115,123,124]. For SRP-dependent secretion, e.g.…”

RECONSTITUTION OF DIFFERENT SIGNAL PEPTIDES FOR ENHANCED THERMOSTABLE ALPHA AMYLASE SECRETION IN Bacillus subtilis

“…Sec pathway is major secretion for approximately 300 proteins in Bacillus genus therefore called the general secretion pathway in this genus [7]. Signal peptide in Sec pathway usually is 18 -40 amino acids long and although the primary structures of different signal peptides show a little similarity and don't have sequence homologous, this signal always consists of three identifiable domains as: a positively charged amino terminal (N-), a central hydrophobic (H-), and carboxyl-terminal (C-) regions [7,8]. N region is 1-5 amino acids long, charged positive, and often has two amino acids Lys and Arg [6,9].…”