Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli.
The catalytic subunit of rabbit skeletal muscle protein phosphatase-1 was expressed in Escherichia coli. Expression of phosphatase-1 in the pET3a vector, which is based on the use of the T7 promoter, resulted in the expression of the enzyme as an insoluble aggregate. The insoluble enzyme could be renatured by high dilutions of the urea-solubilized protein in buffers containing dithiothreitol, Mn2+, and high NaCl concentrations. However, under all conditions tested, only partial (less than 5%) renaturation was achieved. A second attempt was made using a vector with the trp-lac hybrid promoter. In this case it was possible to express the enzyme as a soluble protein at levels of 3-4% of the soluble E. coli protein. The recombinant enzyme was purified by DEAE-Sepharose and heparin-Sepharose chromatography. Approximately 20 mg of purified enzyme was reproducibly obtained from the cells derived from 2 liters of culture. The purified enzyme had a specific activity toward phosphorylase alpha comparable to that reported for the authentic protein and had an Mr of 37,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The recombinant enzyme displayed similar sensitivities to inhibition by inhibitor-2, okadaic acid, and microcystin-LR as for the protein isolated from rabbit muscle. At all stages of purification the recombinant phosphatase behaved as an essentially inactive enzyme that required the presence of microM Mn2+ for full expression of its activity.
Zhang
A J
AJ
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Bai
G
G
Deans-Zirattu
S
S
Browner
M F
MF
Lee
E Y
EY
D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides; D010765:Phosphorylase Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D054645:Protein Phosphatase 1; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D054645:Protein Phosphatase 1; D010765:Phosphorylase Phosphatase