Mechanisms of protein foldingand molecular breeding in halophilic enzymes

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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.