Hepatitis C virus core protein: Carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase
Hepatitis C virus core protein: Carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase
复制标题
DOI：
10.1006/viro.1996.0510
复制
发表时间：
1996-10-01
期刊：
影响因子：
3.7
通讯作者：
Jacobsen, H
中科院分区：
文献类型：
--
作者：
Hussy, P;Langen, H;Jacobsen, H
对丙型肝炎病毒核心蛋白的表达和加工进行了分析。在标准兔网织红细胞裂解物系统和Sf9昆虫细胞中表达的核心蛋白主要有21 kDa(P21)和19 kDa(P19)两条蛋白条带。表达了带有氨基末端组氨酸标签的核心蛋白，这使得Ni2+-NTA柱纯化六组氨酸-P19核心蛋白成为可能。利用质谱仪分析了六组氨酸P19的相对分子质量，并计算出其羧基末端。截短的core/core-E1融合蛋白与小鼠二氢叶酸还原酶(MDHFR)的融合蛋白在预期区域显示切割。切割位点可以通过DHFR-融合伙伴的氨基末端蛋白测序来确定。我们的数据显示，表观分子量约为19 kDa的核心产物不是一个，而是两个，分别终止于氨基酸亮氨酸179或亮氨酸182。这些在丙型肝炎病毒核心的疏水、羧基末端区域的裂解表明：(A)最近提出的真核信号多肽酶(S)(F.Tyke等人)。(1995)J.Biol.化学。19873-19878)。此外，我们的结果表明，这些位点的切割和P19物种的形成不需要在丙型肝炎病毒多聚蛋白的信号酶位点(第191/192位)之前进行处理。(C)1996年学术出版社。
The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine-P19 core with Ni2+-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. tyke et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein. (C) 1996 Academic Press, Inc.