Contribution of Halophilic Nucleoside Diphosphate Kinase Sequence to the Heat Stability of Chimeric Molecule

Abstract

A halophilic nucleoside diphosphate kinase from a moderate halophile, Halomonas sp. 593 (593NDK), was found to be resistant to heat treatment, as indicated by the high level of activity recovery after heating at high temperatures. This is due to reversibility of thermal unfolding, not the high melting temperature, of the protein. The highly homologous NDK from non-halophilic organism, Pseudomonas aeruginosa, showed instability against heat treatment. Chimeric molecules consisting of each half of these two NDKs were constructed and characterized for their heat stability. The results showed that the N-terminal half of 593NDK contributes to the heat stability of the proteins. We discuss the possible reason for the observed difference in resistance to heat treatment between the 593NDK and PaNDK and between two chimeric proteins.

Residue 134 determines the dimer–tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

“…The construction of chimeric proteins from HaNDK and PaNDK was described previously [13]. pET593Pandk encodes chimeric NDK consisting of N‐terminal half of HaNDK followed by C‐terminal half (71th–143th residues) of PaNDK (Ha/Pa‐NDK chimera).…”

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

“…The apparent molecular mass of His-KvSBD12 on SDS-PAGE (14% Laemmli gel) was ∼40 kDa, which was much higher than the true molecular mass, 23 kDa. This slow mobility on SDS-PAGE is one of the characteristics of halophilic proteins [29], due to abnormally weak SDS binding [30]. A single domain KvSBD1 with amino-terminal hexa-His-tag (His-KvSBD1, Fig.…”

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetramericPseudomonascounterpart

“…NDK from various organisms have been studied, including a moderately halophilic bacterium, and purification of NDK from Halomonas sp. 593 (HaNDK) and cloning and expression of HaNDK gene in E. coli with N‐terminal His‐tag have been reported (Yonezawa et al , 2001, 2003; Tokunaga et al , 2006). The database search of primary structures revealed that NDK from P. aeruginosa PAO1 (PaNDK) shows an extensive sequence similarity (78% identity and 89% similarity) to HaNDK.…”