Single-chain Recombinant Human Cytomegalovirus Protease
Single-chain Recombinant Human Cytomegalovirus Protease
复制标题
单链重组人巨细胞病毒蛋白酶
DOI：
--
复制
发表时间：
1995
影响因子：
4.8
通讯作者：
C. Kan
中科院分区：
文献类型：
--
作者：
C. Pinko;Stephen A. Margosiak;Darin Vanderpool;Jeanine C. Gutowski;B. Condon;C. Kan
We report here the production of active recombinant single-chain human cytomegalovirus protease in Escherichia coli and development of a continuous assay for this protease. In order to produce the human cytomegalovirus (HCMV) protease for structural studies and accurate kinetic analysis, mutation of alanine 143 at an internal cleavage site was introduced to prevent autoproteolysis. The resulting soluble 29-kDa A143Q protease was purified to homogeneity as a stable single-chain protein by hydrophobic interaction and ionic-exchange chromatography. The in vivo protein substrate, assembly protein precursor, was also expressed and purified for activity studies. To develop a continuous protease assay, fluorescent synthetic peptide substrates similar to the cleavage sequence P5 to P5′ of the maturation site containing anthranilic acid and nitrotyrosine as a resonance energy transfer donor-acceptor pair were designed. Purified HCMV A143Q protease cleaved the recombinant assembly protein precursor with K and k values of 3.0 ± 1.0 μM and 13.3 ± 1.6 min. The K for peptide substrates is at least 45-fold higher than for the natural protein substrate, but the k values are similar. A sensitive assay was developed using fluorescent peptide substrates, which can detect nM HCMV protease activity.
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
DOI：
10.1073/pnas.88.23.10792
发表时间：
1991-12-01
影响因子：
11.1
作者：
WELCH, AR;WOODS, AS;GIBSON, W
通讯作者：
GIBSON, W