Johnson T M, Mann W R, Dragland C J, Anderson R C, Nemecek G M, Bell P A
Regulatory Toxicology Department, Sandoz Research Institute, Sandoz Pharmaceuticals Corp., East Hanover, New Jersey 07936-1080, USA.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):689-93. doi: 10.1042/bj3090689.
The cDNA encoding rat liver carnitine palmitoyltransferase II (CPT-II) was heterologously expressed using a recombinant baculovirus/insect cell system. Unlike Escherichia coli, the baculovirus-infected insect cells expressed mostly soluble active recombinant CPT-II (rCPT-II). CPT activity from crude lysates of recombinant baculovirus-infected insect cells was maximal between 50 and 72 h post-infection, with a peak specific activity of 100-200 times that found in the mock- or wild-type-infected control lysates. Milligram quantities (up to 1.8 mg/l of culture) of active rCPT-II were chromatographically purified from large-scale cultures of insect cells infected with the recombinant baculovirus. The rCPT-II was found to be: (1) similar in size to the native rat liver enzyme (approximately 70 kDa) as judged by SDS/PAGE; (2) immunoreactive with a polyclonal serum raised against rat liver CPT-II; and (3) not glycosylated. Kinetic analysis of soluble rCPT-II revealed Km values for carnitine and palmitoyl-CoA of 950 +/- 27 microM and 34 +/- 5.6 microM respectively.
使用重组杆状病毒/昆虫细胞系统对编码大鼠肝脏肉碱棕榈酰转移酶II（CPT-II）的cDNA进行了异源表达。与大肠杆菌不同，杆状病毒感染的昆虫细胞主要表达可溶性活性重组CPT-II（rCPT-II）。重组杆状病毒感染的昆虫细胞粗裂解物中的CPT活性在感染后50至72小时达到最大值，其峰值比活性是模拟感染或野生型感染对照裂解物中的100 - 200倍。从感染重组杆状病毒的昆虫细胞大规模培养物中通过色谱法纯化出了毫克量（高达1.8 mg/l培养物）的活性rCPT-II。发现rCPT-II具有以下特点：（1）通过SDS/PAGE判断，其大小与天然大鼠肝脏酶相似（约70 kDa）；（2）与针对大鼠肝脏CPT-II产生的多克隆血清具有免疫反应性；（3）未进行糖基化。对可溶性rCPT-II的动力学分析显示，肉碱和棕榈酰辅酶A的Km值分别为950±27μM和34±5.6μM。
