RRC ID 35616
著者 Yuan S, Xu J, Ge Y, Yan Z, Du G, Wang N.
タイトル Prokaryotic ubiquitin-like ThiS fusion enhances the heterologous protein overexpression and aggregation in Escherichia coli.
ジャーナル PLoS One
Abstract Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.
巻・号 8(4)
ページ e62529
公開日 2013-1-1
DOI 10.1371/journal.pone.0062529
PII PONE-D-13-04045
PMID 23638106
PMC PMC3636215
MeSH Animals Carrier Proteins / chemistry* Carrier Proteins / genetics* Escherichia coli / genetics* Escherichia coli Proteins / chemistry* Escherichia coli Proteins / genetics* Gene Expression Green Fluorescent Proteins / genetics Insulin / genetics Mice Placental Hormones / genetics Protein Folding Protein Multimerization* Protein Stability Protein Structure, Quaternary Recombinant Fusion Proteins / chemistry* Recombinant Fusion Proteins / genetics* Solubility Ubiquitin / chemistry*
IF 2.74
引用数 4
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
原核生物(大腸菌) JW3903-KC ME7970(KY2966)