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 584-128 1 was inserted downstream of the alcohol dehydrogenase promoter and tr ansfected into a haploid strain of yeast. Expression of recombinant CD 45 in yeast reached as high as 5% of the soluble protein. Following re moval of cellular debris by centrifugation and an ammonium sulfate pre cipitation step, the enzyme was purified using phenyl-Sepharose chroma tography, preparative gel filtration, Mono Q anion exchange chromatogr aphy and a final analytical gel fitration step. Enzymatically active m aterial with a purity of greater than or equal to 98% was obtained wit h a yield approaching 50%. The final product gave a K-m of 5.5 mM and a V-max of 87.5 U/mg with p-nitrophenylphosphate and a K-m and V-max o f 0.167 mM and 185 U/mg, respectively, with a phosphotyrosine peptide. The native enzyme purified from Jurkat cells showed comparable K(m)s with both substrates to the recombinant enzyme but displayed substanti ally lower V-max values for both substrates.