TY - JOUR
T1 - Functional expression of soluble forms of human cd38 in escherichia coli and pichia pastoris
AU - Fryxell, Kathryn B.
AU - O’donoghue, Kelly
AU - Graeff, Richard M
AU - Lee, Hon Cheung
AU - Branton, W. Dale
PY - 1995/6
Y1 - 1995/6
N2 - 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.
AB - 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.
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U2 - 10.1006/prep.1995.1043
DO - 10.1006/prep.1995.1043
M3 - Article
C2 - 7663169
AN - SCOPUS:0029310660
SN - 1046-5928
VL - 6
SP - 329
EP - 336
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 3
ER -