FUNCTIONAL EXPRESSION OF SOLUBLE FORMS OF HUMAN CD38 IN ESCHERICHIA-COLI AND PICHIA-PASTORIS

Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nic otinamide adenine dinucleotide (NAD(+)), mobilizes calcium from intrac ellular stores in many cells, The synthesis of cADPR from NAD(+) and i ts subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclas e and a cADPR hydrolase, respectively, The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen C D38, CD38 has been shown to catalyze both the formation and the hydrol ysis of cADPR. In this study, we produced soluble, enzymatically activ e CD38 using recombinant expression techniques in bacteria and yeast, We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids represent ing the putative membrane spanning sequence and short putative intrace llular sequence. For expression in bacteria (Escherichia coli), this c onstruct was cloned into the pFlag-1 plasmid which allows induced, per iplasmic expression and relatively simple purification of the soluble CD38, For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sit es and the resulting construct was expressed as a secreted protein, Bo th systems produce soluble enzymes of approximately 30 kDa and both re combinant enzymes display similar cyclase and hydrolase activities, (C ) 1995 Academic Press, Inc.