Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system.
Human protein disulfide isomerase (PDI) was selected as a fusion partner to construct a gene expression system to enhance the solubility of recombinant protein in Escherichia coli. DREBIII-1, a plant specific transcriptional factor, was found to mainly form inclusion bodies when expressed in either His-tagged or GST-fusion systems in E. coli. In contrast, when fused with PDI, the expressed DREBIII-1 was in a highly soluble and biologically active form. Two fusion proteins, HDP and HPD, were generated by positioning DREBIII-1 at the N-terminal and C-terminal of PDI, respectively. After purification, HDP exhibited a higher stability and showed only one band on SDS-PAGE, while HPD degraded as several bands. HDP was verified to have the biological function of PDI by isomerase activity assay; meanwhile, it also presented the DNA binding and transcriptional activation characteristic of DREBIII-1 in fluorescence quenching and yeast one-hybrid experiments. The PDI fusion expression system was demonstrated to be highly efficient in generating not only soluble but functional desired proteins.
Liu
Yang
Y
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Zhao
Tong-Jin
TJ
Yan
Yong-Bin
YB
Zhou
Hai-Meng
HM
D029688:Brassica napus / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D020218:Response Elements / Q000235:genetics; D012440:Saccharomyces / Q000235:genetics; D012995:Solubility; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019704:Protein Disulfide-Isomerases