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

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(77 4) of the rat alpha 1fsub unit was constructed via polymerase chain re action. The polymerase chain reaction product was successfully subclon ed into the expression vector pET-28 (Novagen), which produces an N-te rminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat alpha-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones mere selected by kanamycin r esistance. Bacterial cultures were grown, and protein synthesis was in duced with isopropyl beta-D-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein (similar to 46 kDa). The fusion protein was affinity-purified from other solubl e cellular proteins via a Ni-NTA column, which routinely yielded simil ar to 20 mg of soluble His(6)-alpha-loop/L cell culture. The His(6)-al pha-loop retained significant native structure, as evidenced by the ab ility of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect agains t chemical modification by either fluorescein isothiocyanate or maleim idylanilinonapthalene sulfonic acid. More specifically, circular dichr oism spectroscopy was used to estimate the secondary structure of the His, loop, revealing an ordered folding composed of 23% alpha-helix, 2 3% antiparallel beta-sheet, 4% parallel beta-sheet, 19% beta-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analo g trinitrophenyl-ATP with high affinity, as determined by measuring th e fluorescence changes associated with binding. Affinities for ATP (si milar to 350 mu M) and ADP (similar to 550 mu M) were determined by th eir ability to compete with and displace 2',3'-O-[2,4,6,-trinitropheny l]-ATP. These nucleotide affinities are similar to those observed for the E-2 conformation of the intact Na,K-ATPase.