title: Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1.
abstract: The genes of the human low Mr 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 and were suitable for high resolution X-ray structure determination.
authors.affiliation: Department of Biochemical Sciences, University of Florence, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Marzocchini
authors.forename: M
authors.initials: M
authors.lastname: Bucciantini
authors.forename: M
authors.initials: M
authors.lastname: Stefani
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: M G
authors.initials: MG
authors.lastname: Thunnissen
authors.forename: P
authors.initials: P
authors.lastname: Nordlund
authors.forename: G
authors.initials: G
authors.lastname: Ramponi
mesh_terms: D003001:Cloning, Molecular; D003461:Crystallography; D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification*; D008970:Molecular Weight; D017027:Protein Tyrosine Phosphatases / Q000302:isolation & purification*; D011994:Recombinant Proteins; D014961:X-Ray Diffraction
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases