Fryxell K B, O'Donoghue K, Graeff R M, Lee H C, Branton W D
Department of Physiology, University of Minnesota, Minneapolis 55455, USA.
Protein Expr Purif. 1995 Jun;6(3):329-36. doi: 10.1006/prep.1995.1043.
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.
环磷酸腺苷二磷酸核糖（cADPR）是烟酰胺腺嘌呤二核苷酸（NAD+）的一种代谢产物，可在许多细胞中从细胞内储存库中动员钙。从NAD+合成cADPR及其随后水解为ADPR分别由ADP-核糖基环化酶和cADPR水解酶催化。从海洋无脊椎动物加州海兔的卵睾体中克隆的ADP-核糖基环化酶与人类淋巴细胞表面抗原CD38具有氨基酸序列同源性。已证明CD38可催化cADPR的形成和水解。在本研究中，我们利用细菌和酵母中的重组表达技术制备了可溶性、具有酶活性的CD38。我们通过切除编码代表推定跨膜序列和短推定细胞内序列的N端氨基酸区域的基因片段，构建了一个编码可溶性CD38形式的基因。为了在细菌（大肠杆菌）中表达，该构建体被克隆到pFlag-1质粒中，该质粒允许诱导性周质表达和相对简单地纯化可溶性CD38。为了在酵母（毕赤酵母）中表达，CD38序列被进一步修饰以消除四个推定的N-糖基化位点，所得构建体作为分泌蛋白表达。这两种系统都产生约30 kDa的可溶性酶，并且两种重组酶都表现出相似的环化酶和水解酶活性。
