Abstract

Enterococcus hirae (E. hirae) vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V_o; NtpI-K₁₀) connected by a central and peripheral stalks. Central stalk of Na⁺-translocating V-type ATPase of E. hirae is composed of NtpC, NtpD and NtpG subunits. The aim of the present study was cloning and expression of these central stalk subunits of E. hirae V-type Na⁺-ATPase. Here we cloned the synthesized DNA fragments, corresponding to ntpC, ntpD and ntpG genes, into the plasmid vector, pET23d. NtpC, NtpD and NtpG subunit proteins were expressed, separately as His-tagged soluble proteins in Escherichia coli BL21(DE3) cells and then, purified by Ni Sepharose 6 fast flow column. Purification of expressed protein was confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). The amount of purified NtpC, NtpD and NtpG subunit proteins were measured as 14, 17 and 15 mg/1 liter culture, respectively.

Key words: Enterococcus hirae, V-ATPase, central stalk subunits, expression.

Abbreviation

PCR, Polymerase chain reaction; DNA, deoxyribonucleic acid;SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; ATP,adenosine 5^-triphosphate; dNTP, deoxynucleotide triphosphate; IPTG, isopropyl (thio) β-D-galactoside; BSA, bovine serum albumin; DTT, dithiothreitol; CBB,Coomassie brilliant blue; EDTA, ethylenediaminetetraacetic acid; LB, Luria-Bertani;m-DM-CA, modified-Davis Mingioli-casamino acid; OD600, optical density at 600 nm.