Ling M H, Qi H Y, Chi C W
Shanghai Institute of Biochemistry, Academia Sinica, China.
J Biol Chem. 1993 Jan 15;268(2):810-4.
Two trypsin inhibitor components of the squash family were isolated and purified from the juice of the towel gourd (Luffa cylindrica) using anhydrotrypsin affinity chromatography followed by high pressure liquid chromatography. The inhibitors were sequenced and found to consist of 28 and 29 amino acid residues. The determined sequences show high similarity to other inhibitors of the squash family, especially in the location of disulfide bonds and the reactive site and also in the COOH-terminal region. A cDNA library of towel gourd was constructed and used as a template for polymerase chain reaction amplification of two cDNA fragments of the inhibitor with an overlapping sequence. A full-length cDNA sequence coding for the inhibitor was then completed. The open reading frame codes for a prepro-inhibitor protein with the pre- and pro-peptides consisting of 21 and 13 residues, respectively. The deduced amino acid sequence of 29 residues for the inhibitor is consistent with that determined by primary structure analysis. The genomic sequence of the mature inhibitor was also ascertained using the total DNA of the towel gourd as a polymerase chain reaction template. The genomic sequence is completely identical with that of the cDNA, showing no intervening sequence.
采用脱水胰蛋白酶亲和层析，随后进行高压液相色谱法，从丝瓜（Luffa cylindrica）汁中分离并纯化出两种南瓜家族的胰蛋白酶抑制剂成分。对这些抑制剂进行测序，发现它们分别由28和29个氨基酸残基组成。所确定的序列与南瓜家族的其他抑制剂具有高度相似性，特别是在二硫键和活性位点的位置以及COOH末端区域。构建了丝瓜的cDNA文库，并将其用作聚合酶链反应扩增具有重叠序列的两种抑制剂cDNA片段的模板。随后完成了编码该抑制剂的全长cDNA序列。开放阅读框编码一种前体原抑制剂蛋白，其前肽和原肽分别由21和13个残基组成。推导的29个残基的抑制剂氨基酸序列与通过一级结构分析确定的序列一致。还以丝瓜的总DNA作为聚合酶链反应模板，确定了成熟抑制剂的基因组序列。该基因组序列与cDNA序列完全相同，未显示出间隔序列。
