Expression and Characterization of the HCV NS3-4A Protein with the Pichia pastoris Expression System and the Baculovirus Expression System

• Main Authors: Huang Yu Wen, 黃鈺雯
• Other Authors: Hwang Lih Hwang
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/36037558592029896316

碩士 === 國立臺灣大學 === 微生物學研究所 === 87 === Hepatitis C virus ( HCV ) is a major etiologic agent of posttransfusional non-A, non-B hepatitis and is highly associated with the development of hepatocellular carcinoma. The virion contains a single-stranded positive-sense RNA genome of about 9.5 kb which encodes a polyprotein of 3010 amino acids with the following order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The NS3 protein of HCV harbors multiple enzymatic activities in its two-domain structure. The N-terminal 181 amino acids of NS3 contains a serine protease activities, while the C terminus of NS3 possesses nucleoside triphosphatase ( NTPase ) and RNA helicase activities. NS4A, forming a stable complex with NS3, plays an important role in the process of NS3 serine protease reaction. In order to obtain full-length NS3/4A for further functional, enzymatic, and structural analyses, in this study, we used Pichia pastoris and baculovirus expression systems to express the NS3/4A protein. In the Pichia pastoris expression system, the recombinant NS3/4A protein had a low expression level. When the nickel-agarose affinity column was used to purify the recombinant NS3/4A protein, a 37 kDa protein always existed, which greatly influenced the purification process. In the baculovirus expression system, the expression of recombinant NS3/4A protein was high. The recombinant protein purified possessed catalytic activities. First, the fusion protein could be autocleaved into NS3 and NS4A, both of which in turn associated to form a stable complex. Second, the NS3/4A protein possessed ATPase and RNA helicase activities；the activities were comparable to those of a truncated NS3 protein containing only the helicase domain. However, the helicase activity of NS3/4A is more resistant to salt , greater than 100 mM in concentration. This fits the true physiologic status more, and may account for the existence of HCV.