Garcia K C, de Sauvage F J, Struble M, Henzel W, Reilly D, Goeddel D V
Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080.
J Biol Chem. 1993 Oct 25;268(30):22397-401.
Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.
鸟苷素是一种由15个氨基酸组成的肽激素，最初从大鼠小肠空肠中分离出来，被证明是肠道热稳定肠毒素受体——鸟苷酸环化酶的内源性激活剂。鸟苷素最初是以一种由115个氨基酸组成的前激素——前鸟苷素的形式合成的，该前激素在一个尚未确定的位点被加工成C端生物活性片段。为了在体外研究前鸟苷素的加工过程，我们通过构建一个表达载体，将其分泌到大肠杆菌的周质空间中，从而大量生成了正确折叠的前激素。然后，通过蛋白酶消化将细菌表达的人源前鸟苷素加工成较小的C端片段。用胰蛋白酶或赖氨酸-C进行消化产生了不同长度的C端肽，这些肽已被纯化和鉴定。鸟苷素-22和鸟苷素-32具有与鸟苷素-15相似的结合亲和力和生物活性，而鸟苷素-63和整个前鸟苷素仅具有最小的生物活性。圆二色光谱显示，前鸟苷素是一种稳定折叠的蛋白质，主要包含β-折叠和β-转角结构。
