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

克隆、测序并在大肠杆菌中表达了嗜热嗜热古生菌KOD1(CpkB)分子伴侣蛋白的β亚基基因。CpkB Geme由1,641个核苷酸组成，编码一个相对分子质量为59,140 Da的蛋白质(546个氨基酸)。用酿酒酵母乙醇脱氢酶(ADH)检测了CpkB对酶稳定性的增强作用。纯化的重组蛋白CpkB可防止ADH的热变性并增强其热稳定性。CpkB在低浓度时需要ATP才能发挥其伴侣功能；然而，当CpkB浓度过高时，CpkB的功能不需要ATP。通过共表达CpkB和CobQ，研究了CpkB在体内对不溶蛋白的增溶作用，表明Cpkb在体内对不溶蛋白具有增溶作用。这些结果表明，β亚基在伴侣活性中起主要作用，并且在没有α亚基的情况下是有功能的。

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.