Mechanisms of protein foldingand molecular breeding in halophilic enzymes

我们首次从皮肤嗜盐杆菌中分离到核苷二磷酸激酶(NDK)，它的稳定性和活性不需要高浓度的盐。这是从极端嗜盐古菌中分离出的酶中第一个显示无盐特性的酶。一步亲和层析法纯化NDK，纯化倍数为540倍。疏水层析法进一步纯化为均一组分。该酶在0~4M的氯化钠存在下很稳定，在2M的氯化钠时酶活最高。结果表明，NDK基因在0和4M的NaCl0和4M时活性最高，约为70%。从皮肤红假单胞菌染色体DNA中克隆了NDK基因，并将其核苷酸序列存放在DDBJ中，登录号为AB036344。在大肠杆菌中表达的Ndkp蛋白定位于可溶性部分，但不具有酶活性。我们发现，表达的酶需要较高的盐浓度才能激活，一旦在高盐存在下激活，酶在去盐后就会变得稳定。这一事实表明，Ndkp不需要高盐来保持稳定性和活性，但确实需要它来进行蛋白质折叠。

We first isolated nucleoside diphsphate kinase (NDK) from Halobacterium cutirubrum which does not require high concentration of salts for its stability and activity. This is first one showing salt-free characteristics among enzymes isolated from extremely halophilic archaea. NDK was highly purified by one step ATP affinity column with 540-fold. It was further purified to homogeneity by hydrophobic chromatography. This enzyme was stable in the presence of 0〜4 M NaCl, and showed maximum activity at 2 M NaCl. It showed about 70 % of maximum activity at 0 and 4 M NaCl.The ndk gene was cloned and sequenced from H, cutirubrum chromosomal DNA and its nucleotide sequence has been deposited to DDBJ with accession number of AB036344.We have constructed the expression vector for this gene in E. coli. The expressed Ndkp in E. coli was localized in soluble fraction but did not have enzyme activity. We found that the high salt concentration was required for the activation of this expressed enzyme, and once enzyme was activated in the presence of high salt, it was stable after removal of salt. This fact indicated that Ndkp does not require high salt for stability and activity, but does require it for the protein folding.