Lucas N, Mazaud-Aujard C, Bremaud L, Cenatiempo Y, Julien R
Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Eur J Biochem. 1994 Jun 1;222(2):247-54. doi: 10.1111/j.1432-1033.1994.tb18863.x.
An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of kappa-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein.
通过一系列色谱方法，将黄色粘球菌DK101在营养生长期间分泌的一种酸性内切蛋白酶（MAEP）纯化至同质。该内切蛋白酶在酸性条件（pH 5.9）下切割κ-酪蛋白的苯丙氨酸-甲硫氨酸键。其表观分子量和等电点估计分别为12 kDa和4.5。根据N端氨基酸序列，设计了一组用于聚合酶链反应的引物。相应DNA片段（84 bp）的扩增产生了一个探针，然后用于筛选黄色粘球菌的表达DNA文库并分离出一个含有2127 bp插入片段的重组质粒。核苷酸序列包含一个585个核苷酸的开放阅读框（ORF），编码195个氨基酸，与纯化的MAEP的N端氨基酸序列具有高度相似性。从该ORF推断的多肽序列表明，成熟酶应由195个氨基酸的前体蛋白产生的131个氨基酸组成。
