High level expression and purification of the enzymatically active cytoplasmic region of human CD45 phosphatase from yeast.
The cytoplasmic region of human CD45 corresponding to residues 584-1281 was inserted downstream of the alcohol dehydrogenase promoter and transfected into a haploid strain of yeast. Expression of recombinant CD45 in yeast reached as high as 5% of the soluble protein. Following removal of cellular debris by centrifugation and an ammonium sulfate precipitation step, the enzyme was purified using phenyl-Sepharose chromatography, preparative gel filtration, Mono Q anion exchange chromatography and a final analytical gel filtration step. Enzymatically active material with a purity of > or = 98% was obtained with a yield approaching 50%. The final product gave a Km of 5.5 mM and a Vmax of 87.5 U/mg with p-nitrophenylphosphate and a Km and Vmax of 0.167 mM and 185 U/mg, respectively, with a phosphotyrosine peptide. The native enzyme purified from Jurkat cells showed comparable Kms with both substrates to the recombinant enzyme but displayed substantially lower Vmax values for both substrates.
Department of Immunopharmacology, Sterling Winthrop Pharmaceutical Research Division, Collegeville, PA 19426-0900.
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Pacitti
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Stevis
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Evans
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Trowbridge
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Higgins
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line / Q000201:enzymology; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000814:Aniline Compounds; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C068798:4-aminophenylphosphate; D017027:Protein Tyrosine Phosphatases