[Intrinsic prokaryotic promoter activity of SUMO gene and its applications in the protein expression system of Escherichia coli].
Nowadays, SUMO fusion system is important for recombinant protein production in Escherichia coli, yet a few aspects remain to be improved, including the efficacy for vector construction and protein solubility. In this study, we found the SUMO gene Smt3 (Sm) of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning, and the gene coding regions of SUMOs in most species had a sigma70-dependent prokaryotic promoter embedded, through the prediction via the BPROM program developed by Softberry. By combining the characters of Sm promoter activity and the Stu I site (added at the 3'-terminal of Sm), and introducing a His-tag and a hyper-acidic solubility-enhancing tag, we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm'-LacZa fusion gene. Experimentally started from these vectors, several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis. The results manifest a few of expectable advantages such as rapid vector construction, highly soluble protein expression and feasible co-expression of correlated proteins. Conclusively, our optimized SUMO fusion technology herein could confer a large potential in E. coli protein expression system, and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.
College of Agronomy, Shandong Agricultural University, Tai'an 271000, China.
Yanhong
Y
Qi
Zhurong
Z
Zou
Huaying
H
Zou
Yunliu
Y
Fan
Chunyi
C
Zhang
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis / Q000235:genetics*; D058207:Sumoylation
D011993:Recombinant Fusion Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins