Functional recombinant rabbit muscle phosphoglucomutase from Escherichia coli.
The gene coding for phosphoglucomutase (PGM) from Oryctolagus cuniculus (rabbit) has been expressed in Escherichia coli under a T7 expression system with a His-tag. About half of the expressed PGM protein was present in inclusion bodies, but this protein was inactive when solubilized. The protein in the soluble cell fraction was isolated and purified in one step on a Ni-NTA column. The eluate from this column was adjusted to 95% saturated ammonium sulfate, and the resulting protein precipitate was resuspended in sodium phosphate buffer and dialyzed against 2.5 M ammonium sulfate. The final yield of protein was about 10 mg per liter of LB medium. The protein was judged to be greater than 90% pure on the basis of gel electrophoresis and activity measurements (128 U per milligram). Our motivation for developing this bacterial production system for PGM has been to prepare sufficient quantities of stable-isotope-labeled protein for experiments that utilize recently developed NMR technologies suitable for proteins the size of PGM (61.6 kDa). Preliminary NMR studies indicate that the current level of purity is adequate for this work. The construct described here was designed to incorporate an N-terminal His-tag for ease of isolation. Although PGM is a metalloprotein, the His-tag does not appear to interfere with activity. The presence of the His-tag should not pose a problem for proposed (31)P NMR investigations of the protein and its complexes in aqueous solution or incorporated into reverse micelles. However, we plan to design a cleavable His-tag for later (1)H, (13)C, (15)N studies of the active site, which includes essential histidine residues.
Chae
Y K
YK
Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Markley
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010733:Phosphoglucomutase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D010733:Phosphoglucomutase