Facilitated folding and subunit assembly in Escherichia coli and in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag.
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 degrees 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.
Ishibashi
Matsujiro
M
Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
Arakawa
Tsutomu
T
Tokunaga
Masao
M
D001105:Archaea / Q000378:metabolism*; D002455:Cell Division; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006358:Hot Temperature; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012492:Salts / Q000494:pharmacology; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
D003432:Cross-Linking Reagents; D012492:Salts; D012965:Sodium Chloride; D009701:Nucleoside-Diphosphate Kinase; D013917:Thrombin