High-level expression of the C-terminal hydrophobic region of HCV E2 protein ectodomain in E. coli.
High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484-622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567-700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568-571 of E2. C-terminal region of E2 ectodomain (aa 567-730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.
Liu
Jing
J
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, People's Republic of China.
Kong
Yuying
Y
Zhu
Lixin
L
Wang
Yuan
Y
Li
Guangdi
G
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D018937:Hepatitis C Antibodies; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000235:genetics
D018937:Hepatitis C Antibodies; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus