Facilitated folding and subunit assembly inEscherichia coliand in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag

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Abstract
We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa‐His‐tag at amino‐terminus of HsNDK (His‐HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His‐HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His‐HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino‐terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino‐terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.