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.
Pacitti
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A
Department of Immunopharmacology, Sterling Winthrop Pharmaceutical Research Division, Collegeville, PA 19426-0900.
Stevis
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P
Evans
M
M
Trowbridge
I
I
Higgins
T J
TJ
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