In Vitro Stabilization and In Vivo Solubilization of Foreign Proteins by the beta Subunit of a Chaperonin

克隆了嗜热古细菌焦球菌（Pyrococcus sp.）菌株KOD1 （cpkB）分子伴侣蛋白β亚基的编码基因，对其进行了测序，并在大肠杆菌中进行了表达。cpkB基因组由1,641个核苷酸组成，编码一个分子质量为59,140 Da的蛋白质（546个氨基酸）。利用酿酒酵母醇脱氢酶（ADH）检测了CpkB对酶稳定性的增强作用。纯化的重组CpkB可防止ADH热变性，提高ADH的热稳定性。在低CpkB浓度下，CpkB需要ATP才能发挥伴侣蛋白的功能；然而，当过量存在时，CpkB在没有ATP的情况下发挥作用。通过共表达CpkB和coq (cobryic acid synthase)，研究了体内伴侣蛋白对不溶性蛋白的增溶功能，表明CpkB对体内不溶性蛋白的增溶有用。这些结果表明β亚基在伴侣蛋白活性中起主要作用，并且在没有α亚基的情况下发挥功能。

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp.strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB geme is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme sstability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinat CpkB prevents thermal denaturation and enhances thermostability of ADH.CpkB requires ATP for its chaperonin function at a low CpkB concentration ; however, CpkB functions without ATP when present in excess. In vivo chapernoin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indication that Cpkb is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subnit plays a major role in chaperonin acitivity and is functional without the alpha subunit.