The M4M5 Cytoplasmic Loop of the Na,K-ATPase, Overexpressed in Escherichia coli, Binds Nucleoside Triphosphates with the Same Selectivity as the Intact Native Protein (original) (raw)

1998, Journal of Biological Chemistry

Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys 354 -Lys 774 of the rat ␣1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat ␣-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl ␤-Dthiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein (ϳ ϳ46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded ϳ ϳ20 mg of soluble His 6 -␣-loop/L cell culture. The His 6 -␣-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His 6 loop, revealing an ordered folding composed of 23% ␣-helix, 23% antiparallel ␤-sheet, 4% parallel ␤-sheet, 19% ␤-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP (ϳ350 M) and ADP (ϳ ϳ550 M) were determined by their ability to compete with and displace 2,3-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E 2 conformation of the intact Na,K-ATPase.