TY - JOUR
T1 - Increase of soluble expression in Escherichia coli cytoplasm by a protein disulfide isomerase gene fusion system
AU - Liu, Yang
AU - Zhao, Tong Jin
AU - Yan, Yong Bin
AU - Zhou, Hai Meng
N1 - Funding Information:
This work was supported by funds from the National Key Science and Technology Item of China (No. 2002AA224091), the National Key Basic Research Specific Foundation of China (No. G 1999075607), the China Natural Science Foundation (No. 30221003 and No. 60401009), and the Basic Research Fund (JC2003061) and the 985 Project of Tsinghua University.
PY - 2005/12
Y1 - 2005/12
N2 - 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.
AB - 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.
KW - Fusion protein
KW - Inclusion bodies
KW - Protein disulfide isomerase
KW - Soluble expression and purification
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U2 - 10.1016/j.pep.2005.03.030
DO - 10.1016/j.pep.2005.03.030
M3 - Article
C2 - 15882951
AN - SCOPUS:27644571313
SN - 1046-5928
VL - 44
SP - 155
EP - 161
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -