Expression of recombinant inhibitor-2 in E. coli and its utilization for the affinity chromatography of protein phosphatase-1.
The coding sequence for rabbit muscle protein phosphatase inhibitor-2 was inserted into the pET3a expression vector. This vector allowed the expression of recombinant inhibitor-2 at levels of ca. 1.5% of the soluble protein. A simple procedure allowed the purification of inhibitor-2 from Escherichia coli lysates. This involved heat-treatment, followed by chromatography on Blue-Sepharose and Q-Sepharose. Recombinant inhibitor-2 inhibited rabbit muscle protein phosphatase-1 with a potency similar to that reported for the wild type protein. The recombinant protein was coupled to CH-Sepharose and this support was found to bind the catalytic subunit of protein phosphatase-1 with high efficiency. A procedure for a single-step affinity purification of recombinant ppase-1 from E. coli lysates was shown to be feasible.
Zhang
Z
Z
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Zhao
S
S
Zirattu
S D
SD
Bai
G
G
Lee
E Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis*; D009132:Muscles / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D054645:Protein Phosphatase 1; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D000097847:Protein Phosphatase Inhibitory Proteins
D017931:DNA Primers; D009124:Muscle Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011506:Proteins; D011994:Recombinant Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins