Cloning, expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp. #593

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

“…Residual Activity after Heat-Treatment, Optimum Reaction Temperature and CD Measurement of 593NDK 593NDK was purified by ATP-affinity column in low salt buffer, indicating that the enzyme does bind its substrate, ATP, and hence is in the native structure in low salt buffer [4,5]. The heat stability of the enzyme in low salt buffer (0.2 M NaCl in 50 mM Tris-HCl buffer, pH8.0) was studied after incubating the enzyme at various temperatures for 5 min and cooling it on ice.…”

“…593 and P. aeruginosa PAO1 was described in previous paper [5]. The construction of pET593ndk for the expression of His-593NDK in E. coli was also reported before [5].…”

“…593 was grown in Nutrient broth medium (1 % beef extract and 1 % polypeptone), pH7.0, containing 2 Purification of NDKs from Halomonas sp. 593 and from P. aeruginosa 593NDK was purified by ATP affinity column chromatography as described [4,5]. Pseudomonas aeruginosa cells suspended in 50 mM Tris-HCl buffer, pH8.0, containing 2 mM MgCl 2 and 0.15 M NaCl were disrupted by sonication and soluble fraction after centrifugation of crude homogenate was subjected to ATP-agarose column chromatography.…”

“…593 and P. aeruginosa PAO1 was described in previous paper [5]. The construction of pET593ndk for the expression of His-593NDK in E. coli was also reported before [5]. For the construction of pETPandk for the expression of His-PaNDK in E. coli, ndk from P. aeruginosa was amplified using a forward primer (5'-AGATATACATATGGCACTGCAACG CAC : NdeI site was underlined) and a reverse primer (5'-CCCTCGAGTCAGCGAATGCGCT : XhoI site was underlined).…”

“…We have been studying enzymes and proteins derived from halophilic organisms, including DnaK and nucleoside diphosphate kinase (HsNDK) from extremely halophile, Halobacterium salinarum [1][2][3] and nucleoside diphosphate kinase, β-lactamase (βLA), porin and HrdC from moderate halophiles [4][5][6][7][8][9]. One of the emerging characteristics of the proteins from these halophilic organisms is its highly efficient refolding from chemically and thermally unfolded state [3,6,7].…”