牛乳酸脱氢酶-A同工酶的一级结构及其在大肠杆菌中的合成

Primary structure of bovine lactate dehydrogenase-A isozyme and its synthesis in Escherichia coli.

作者信息

Ishiguro N, Osame S, Kagiya R, Ichijo S, Shinagawa M

机构信息

Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Japan.

出版信息

Gene. 1990 Jul 16;91(2):281-5. doi: 10.1016/0378-1119(90)90101-v.

DOI:10.1016/0378-1119(90)90101-v

PMID:2210387

Abstract

Eleven cDNA clones encoding lactate dehydrogenase(LDH)-A isozyme were isolated from a bovine lymphocyte cDNA library, and the nucleotide sequences of three of the clones (pLDH5, pLDH9 and pLDH12) were determined. With the exception of variation in the 5' portion, two cDNA clones (pLDH9 and pLDH12) appeared to contain the full-length cDNA of 1786 bp, consisting of the protein-coding sequence (996 bp), the 5'- and the 3'-untranslated regions and the poly(dA) tail. The predicted amino acid (aa) sequence of bovine LDH-A (332 aa) showed 96.7% homology with that of pig LDH-A. The protein-coding cDNA region (1650 bp) was inserted into an Escherichia coli expression vector ptac11 and expressed. The protein synthesized in E. coli showed enzyme activity of LDH and was identified by cellogel electrophoresis as LDH-5 isozyme whose subunit M chain is the product of the LDH-A gene.

摘要

从牛淋巴细胞cDNA文库中分离出11个编码乳酸脱氢酶（LDH）-A同工酶的cDNA克隆，并测定了其中3个克隆（pLDH5、pLDH9和pLDH12）的核苷酸序列。除5'端部分存在差异外，两个cDNA克隆（pLDH9和pLDH12）似乎包含1786 bp的全长cDNA，由蛋白质编码序列（996 bp）、5'和3'非翻译区以及聚（dA）尾组成。牛LDH-A的预测氨基酸（aa）序列（332个aa）与猪LDH-A的序列显示出96.7%的同源性。将蛋白质编码cDNA区域（1650 bp）插入大肠杆菌表达载体ptac11并进行表达。在大肠杆菌中合成的蛋白质表现出LDH的酶活性，并通过细胞凝胶电泳鉴定为LDH-5同工酶，其亚基M链是LDH-A基因的产物。