Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli

Abstract

Escherichia coli is the best-established microbial host strain for production of proteins and chemicals, but has a weakness for not secreting high amounts of active heterologous proteins to the extracellular culture medium, of which origins belong to whether prokaryotes or eukaryotes. In this study, Candida antarctica lipase B (CalB), a popular eukaryotic enzyme which catalyzes a number of biochemical reactions and barely secreted extracellularly, was expressed functionally at a gram scale in culture medium by using a simple amino acid-tag system of E. coli. New fusion tag systems consisting of a pelB signal sequence and various anion amino acid tags facilitated both intracellular expression and extracellular secretion of CalB. Among them, the N-terminal five aspartate tag changed the quaternary structure of the dimeric CalB and allowed production of 1.9 g/L active CalB with 65 U/mL activity in culture medium, which exhibited the same enzymatic properties as the commercial CalB. This PelB-anion amino acid tag-based expression system for CalB can be extended to production of other industrial proteins hardly expressed and exported from E. coli, thereby increasing target protein concentrations and minimizing purification steps.

Application of repeated aspartate tags to improving extracellular production of Escherichia coli l-asparaginase isozyme II

“…Although the protein band of the secreted AnsB for the case of P-D3-AnsB was also thicker than that for P-AnsB, specific activities of P-AnsB and P-D3-AnsB in the culture medium were almost identical. We postulated that attachment of the tags to the N-terminal end of AnsB might alter the protein activity as previously reported [17,20], and hence, result in similar specific activities in medium.…”

“…We have previously shown that the GSP machinery specifically exported the dimer form of D5-CalB [17]. However, a previous study reported that the gsp genes are transcriptionally silent in the E. coli K-12 strain [23].…”

“…In our previous report, recombinant Candida antarctica lipase B (CalB) with the N-terminal 5-aspartate tag dramatically increased both expression and secretion of CalB with the aid of the pelB signal sequence in recombinant E. coli [17]. These facts suggest that an adequate alteration of the N-terminal end of AnsB by fusion of anion amino acids could improve soluble expression and transport efficiency across the membranes as done for recombinant CalB.…”

“…We hypothesized that the gsp genes encoding a putative secreton would be a factor specifically affecting transport efficiency of the D5-AnsB across the outer membrane as observed for D5-CalB. Based on this hypothesis, expression vectors of pET-26b(+)-P-AnsB and pET-26b(+)-P-D5-AnsB were transformed to gspDE [17] lacking a part of the 15 gsp genes coding for all components of the GSP secreton. To assess the effects of destruction of a part of the GSP secreton on recombinant AnsBs expression, batch fermentations of the gspDE/P-AnsB and gspDE/P-D5-AnsB strains were performed, and the crude proteins were subjected to AnsB activity assay (Fig.…”

“…The chromosomal DNA of E. coli K-12 was used as the source of the AnsB gene. A gspDE-deficient background strain (designated here as gspDE) lacking a part of the fifteen gsp genes was previously constructed [17].…”