Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris.
Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase 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 CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active 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 representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic 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 sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.
Fryxell
K B
KB
Department of Physiology, University of Minnesota, Minneapolis 55455, USA.
O'Donoghue
K
K
Graeff
R M
RM
Lee
H C
HC
Branton
W D
WD
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D036563:Cyclic ADP-Ribose; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis
D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1