Liao J H, Hung C C, Lee J S, Wu S H, Chiou S H
Laboratory of Crystallin Research, National Taiwan University, Taipei.
Biochem Biophys Res Commun. 1998 Mar 6;244(1):131-7. doi: 10.1006/bbrc.1998.8226.
alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species. Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins. In order to compare this chaperone-like structural protein from the eye lenses of different mammalian species, we have cloned and expressed one of the main alpha-crystallin subunits, i.e., alpha B crystallin, from the porcine lenses in order to facilitate the structure-function evaluation and comparison of this chaperonin protein. cDNA encoding alpha B subunit chain was obtained using a new "Marathon cDNA amplification" protocol of Polymerase Chain Reaction (PCR). PCR-amplified product corresponding to alpha B subunit was then ligated into pGEM-T plasmid and prepared for nucleotide sequencing by the dideoxy-nucleotide chain-termination method. Sequencing several positive clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 525 base pairs similar to human and bovine alpha B subunits, covering a deduced protein sequence of 175 amino acids including the universal translation-initiating methionine. The porcine alpha B crystallin shows only 3 and 7 residues difference to bovine and human alpha B crystallins respectively, revealing the close relatedness among mammalian eye lens proteins. The sequence differences between porcine and sub-mammalian species such as chicken and bullfrog are much greater, especially at the N- and C-terminal regions of these alpha B crystallins. Expression of alpha B subunit chain in E. coli vector generated a polypeptide which can cross-react with the antiserum against the native and purified alpha B subunit from the native porcine lenses albeit with a much lower activity.
α-晶状体蛋白是所有脊椎动物晶状体中存在的一种主要晶状体蛋白。最近的研究表明，牛α-晶状体蛋白具有与小热休克蛋白相似的真正伴侣活性。为了比较来自不同哺乳动物物种眼晶状体的这种类似伴侣的结构蛋白，我们克隆并表达了猪晶状体中主要的α-晶状体蛋白亚基之一，即αB晶状体蛋白，以便于对这种伴侣蛋白进行结构-功能评估和比较。使用聚合酶链反应（PCR）的新“马拉松cDNA扩增”方案获得了编码αB亚基链的cDNA。然后将对应于αB亚基的PCR扩增产物连接到pGEM-T质粒中，并通过双脱氧核苷酸链终止法进行核苷酸测序。对几个含有编码αB晶状体蛋白亚基DNA插入片段的阳性克隆进行测序，构建了一个仅525个碱基对的完整全长阅读框，与人及牛的αB亚基相似，涵盖了一个推导的175个氨基酸的蛋白质序列，包括通用的翻译起始甲硫氨酸。猪αB晶状体蛋白与牛和人αB晶状体蛋白分别仅相差3个和7个残基，揭示了哺乳动物眼晶状体蛋白之间的密切相关性。猪与鸡和牛蛙等亚哺乳动物物种之间的序列差异要大得多，尤其是在这些αB晶状体蛋白的N端和C端区域。αB亚基链在大肠杆菌载体中的表达产生了一种多肽，该多肽可以与针对天然猪晶状体中天然和纯化的αB亚基的抗血清发生交叉反应，尽管活性要低得多。
