Brown N F, Sen A, Soltis D A, Jones B, Foster D W, McGarry J D
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):79-86. doi: 10.1042/bj2940079.
cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5' end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3' to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.
发现与大鼠肉碱棕榈酰转移酶II（CPT II）前体和成熟形式相对应的cDNA能在大肠杆菌中轻松表达。在这两种情况下，都产生了具有催化活性的免疫反应性蛋白，并且该蛋白在很大程度上与膜相关。该酶的前体形式未经过蛋白水解加工。从cDNA编码区的5'端去除126 bp可使截短的CPT II（缺少成熟蛋白的N端17个残基）表达，但该产物无活性。编码人CPT II前体和成熟形式的cDNA在大肠杆菌中无法直接表达。然而，当将后者的cDNA与编码谷胱甘肽S-转移酶的序列框内连接到3'端时，这一障碍被克服了。该构建体产生了大量相应的融合蛋白，其中一部分是可溶的且具有催化活性。对各种cDNA进行体外转录和翻译表明，大鼠CPT II在SDS/PAGE上的迁移率低于其人类对应物（尽管它们的氨基酸数量相同）是蛋白质自身一级序列的固有特性。此外，还发现人cDNA含有一个T3 RNA聚合酶的人为终止信号，而T7聚合酶可以绕过该信号。因此，大鼠CPT II可以在大肠杆菌中以活性形式表达，其特性与线粒体中的酶相似，这为今后确定该分子内的活性位点开辟了道路。对于人CPT II的类似研究将需要另一种表达系统。
