Expression, purification and preliminary crystal analysis of the human low M_r phosphotyrosine protein phosphatase isoform 1

Abstract

The genes of the human low M r phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S-transferase. The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X-ray-grade, diffracted to better than 2.4 A î and were suitable for high resolution X-ray structure determination.z 1998 Federation of European Biochemical Societies.

The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcγRIIA-mediated cell activation

“…Rabbit polyclonal anti-LMW-PTP antibody was prepared as previously described. 30 Human recombinant LMW-PTP was expressed in E coli, and purified as previously described 31 ; 1U defines the amount of enzyme able to hydrolyze 1 mol p-nitrophenyl phosphate in 1 minute at 37°C and pH 5.5. Phosphotyrosine monoclonal antibody 4G10, anti-LAT, and anti-FcR ␥-chain polyclonal antibodies were from Upstate Biotechnology (Lake Placid, NY).…”

New 4-[(5-arylidene-2-arylimino-4-oxo-3-thiazolidinyl)methyl]benzoic acids active as protein tyrosine phosphatase inhibitors endowed with insulinomimetic effect on mouse C2C12 skeletal muscle cells

“…Enzyme expression and purification were achieved in the Escherichia coli TB1 strain [71]. Briefly, the recombinant fusion proteins were purified from bacterial lysate using a single step affinity chromatography on glutathione-Sepharose.…”

Low molecular weight protein tyrosine phosphatase (LMW-PTP) and its possible physiological functions of redox signaling in the eye lens

“…Furthermore, we confirmed that all three isoforms of LMW-PTP generated from one single gene by alternative splicing were present in HLE B3 cells. Both isoforms LMW-PTP1 and LMW-PTP2 were identical to those cloned from human RBC [9], placenta [23] and Jurkat T leukemia cells [10]. The third isoform LMW-PTP3 was identical to the smaller protein found in T cells [10] but different from the one isolated from the placenta [23].…”