[Expression and kinetic analysis of catalytic domain of protein tyrosine phosphatases SHP-1/SHP-2].
In order to express and purify the catalytic domain of SHP-1/SHP-2 (named as D1C and D2C respectively) and determine their kinetics, the constructed D1C and D2C plasmids were transformed into Escherichia coli BL21 and the expression was induced with IPTG. The harvested cells were suspended in extraction buffer. After sonication, the solution was applied to HPLC and the results were confirmed by SDS-PAGE. The purified peptides were further subjected to kinetic specificity study using synthetic phosphotyrosine (pY) as substrate by malachite green method and analyzed by Lineweaver-Burk plot calculation. From this study, we found D1C and D2C were expressed successfully in soluble state in Escherichia coli BL21 and purified efficiently with HPLC system. The molecular weight of D1C was 34.6 kD, and its Michaelis constant (K(m)) was 2.04 mmol catalytic constant (K(cat)) was 44.98 s(-1), specific constant (K(cat)/K(m)) was 22.05 L/(mmol x s); the molecular weigh of D2C was 35.3 kD, and its Michaelis constant (K(m)) was 2.47 mmol, catalytic constant (K(cat)) was 27.45 s(-1), specific constant (K(cat)/K(m)) was L/(mmol x s). The enzyme activity of D1C is stronger than that of D2C.
Mo
Yi
Y
College of Animal Science and Technology, Guangxi University, Nanning 530004, China.
Wang
Wei
W
Liang
Fangfang
F
Fu
Guanyuan
G
Jiang
Hesheng
H
Zhou
Wayne
W
D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6