鲤肌肉肌原纤维结合型丝氨酸蛋白酶的分子克隆

肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。

Molecular cloning of a myofibril-bound serine proteinase (MBSP) from common carp (Cyprinus carpio) muscle

Myofibril-bound serine proteinase (MBSP) is a recently identified serine proteinase, which is responsible for myofibrillar protein degradation and gel softening in the preparation of fish cakes. However, the full-length sequence of MBSP has never been reported. In the present study, degenerate primers were designed according to the N-terminal amino acid sequence of carp MBSP and well-conserved active site motif of serine proteinases. Based on RT-PCR and PCR amplification of the cDNA fragment from N-terminal to active site motif and rapid amplification of cDNA end (RACE) of the 3′- and 5′- regions, the full-length cDNA of MBSP was confirmed. Analysis of the nucleotide sequence of carp MBSP revealed that the cDNA clone has an open reading frame of 732 bp encoding a protein of 243 amino acid residues and a signal peptide of 21 amino acid residues. Three residues (His61, Asp107 and Ser197) forming the typical catalytic triad of serine proteinases for functional activity were conserved in the polypeptide sequence. Mature MBSP contains 222 amino acid residues with an estimated molecular weight of about 24.5 ku, which is smaller than that of its native protein (30 ku). The estimated pI of mature MBSP is 10.43. Sequence alignment showed that carp MBSP has identity of 80.6% to crucian carp MBSP, 55.8% to porcine trypsin, 55.3% to bovine trypsin, 53.9% to flounder trypsin and 39.2% to a chymotrypsin type serine proteinase mekratin, which is from the skeletal muscle of hamster. The high content (11.93%) of Lys residue distinguished carp MBSP from other serine proteinases and this may account for its myofibril-binding characteristic.