Nucleotide diphosphate kinase (NDK) was purified from 12 strains of halobacteria using ATP-agarose chromatography and their N-terminal amino acid sequences were determined. The electrophoretic mobilities of these enzymes differed significantly on the native-PAGE or the SDS-PAGE when the samples were not heat treated. Comparison of seven complete amino acid sequences from seven species of Haloarcula_and those from Har. californiae and Har. japonica, whose N-terminal 3 amino acids have not been determined yet, revealed that they are very similar and differed at only 1 to 4 residues in 153 residues. NDKs from Haloarcula quadrata and Har. sinaiiensis differed only at the 30th amino acid (arginine vs. cysteine), yet they showed a remarkable difference in their salt-response patterns, suggesting that a single amino acid substitution can cause a one molar shift in the optimal NaCl concentration.

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Genes annotated to threonine dehydrogenase, α-mannosidase, and glutamate dehydrogenase of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were used to test the expression system of the thermophilic eubacterium Thermus thermophilus HB27. Using the previously described P215 promoter, the three genes were successfully expressed, but at significantly lower levels than in the Escherichia coli expression system with the T7 promoter. Replacement of the promoter region with P31 or Pslp_promoter improved the expression to a level comparable to or exceeding that in the E. coli system. Notably, α-mannosidase activity was clearly detected with the P31 and Pslp_promoters, which could not be detected in the E. coli system. Moreover, with 6 x His-tag fusions of these enzymes at their COOH- or NH₂-termini, these proteins could be detected in the crude extracts of T. thermophilus by Western blotting, indicating that the increment of each enzyme activity was actually the result of enzyme production. These results demonstrate that the host-vector system of T. thermophilus is useful for expression of genes from hyperthermophiles.

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