Xu W F, Tao W K, Gong Z Z, Chi C W
Shanghai Institute of Biochemistry, Academia Sinica, China.
J Biochem. 1993 Feb;113(2):153-8. doi: 10.1093/oxfordjournals.jbchem.a124019.
Arrowhead double-headed proteinase inhibitors A and B consist of 150 amino acid residues with three disulfide bonds. Based on their primary structures, three cDNA fragments of the inhibitors were amplified in vitro by the PCR method using a constructed arrowhead cDNA library as a template. With the overlapping sequences, the full-length cDNA sequences of the inhibitors were then ascertained. The open reading frame encodes a pre-inhibitor, including a 24 residue signal peptide. The deduced amino acid sequences are consistent in principle with those determined by primary structure analysis, except that there are seven extra residues in the C-terminal part of the inhibitors, which might be cleaved off by proteinase post-processing immediately after protein synthesis. It is worth pointing out that cDNAs of both inhibitors A and B contain an 87 bp intron in the AAG codon of residue Lys-97. According to the elucidated cDNA sequences, the structural genes of inhibitors A and B were amplified using the total cDNA or genomic DNA of arrowhead as a PCR template. It was indicated that both the cDNA and genomic structures of inhibitors A and B have the same sequences.
慈菇双头蛋白酶抑制剂A和B由150个氨基酸残基组成，含有三个二硫键。根据它们的一级结构，以构建的慈菇cDNA文库为模板，通过PCR方法在体外扩增出抑制剂的三个cDNA片段。利用重叠序列，随后确定了抑制剂的全长cDNA序列。开放阅读框编码一个前体抑制剂，包括一个24个残基的信号肽。推导的氨基酸序列原则上与通过一级结构分析确定的序列一致，只是在抑制剂的C末端部分有七个额外的残基，这些残基可能在蛋白质合成后立即被蛋白酶后加工切除。值得指出的是，抑制剂A和B的cDNA在第97位赖氨酸残基的AAG密码子中都含有一个87 bp的内含子。根据阐明的cDNA序列，以慈菇的总cDNA或基因组DNA为PCR模板，扩增出抑制剂A和B的结构基因。结果表明，抑制剂A和B的cDNA和基因组结构具有相同的序列。
