Molecular cloning and expression in yeast of caprine prochymosin

被引：26

作者：

Vega-Hernánez, MC ^{[1
]}

Gómez-Coello, A ^{[1
]}

Villar, J ^{[1
]}

Claverie-Martín, F ^{[1
]}

机构：

[1] Nuestra Senora Candelaria Univ Hosp, Mol Biol Lab, Res Unit, Santa Cruz de Tenerife 38010, Spain

来源：

JOURNAL OF BIOTECHNOLOGY | 2004年 / 114卷 / 1-2期

关键词：

caprine prochymosin; Chymosin; aspartic proteinase; milk clotting; Capra hircus;

D O I：

10.1016/j.jbiotec.2004.06.002

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

We cloned and characterized a preprochymosin cDNA from the abomasum of milk-fed kid goats. This cDNA contained an open reading frame that predicts a polypeptide of 381 amino acid residues, with a signal peptide and a proenzyme region of 16 and 42 amino acids, respectively. Comparison of the caprine preprochymosin sequence with the corresponding sequences of lamb and calf revealed 99 and 94% identity at the amino acid level. The cDNA fragment encoding the mature portion of caprine prochymosin was fused in frame both to the killer toxin signal sequence and to the alpha-factor signal sequence-FLAG in two different yeast expression vectors. The recombinant plasmids were transformed into Kluyveromyces lactis and Saccharomyces cerevisiae cells, respectively. Culture supernatants of both yeast transformants showed milk-clotting activity after activation at acid pH. The FLAG-prochymosin fusion was purified from S. cerevisiae culture supernatants by affinity chromatography. Proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed K-casein. (C) 2004 Elsevier B.V. All rights reserved.

引用

页码：69 / 79

页数：11

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