Kislitsyn Iu A, Rebrikov D V, Dunaevskiĭ Ia E, Rudenskaia G N
Chemical Faculty, Moscow State University, Vorob'evy gory, Moscow, 119899 Russia.
Bioorg Khim. 2003 May-Jun;29(3):269-76. doi: 10.1023/a:1023932415680.
Trypsin from hepatopancreas of the Paralithodes camtschaticus crab was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. The cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the Penaeus vannamei shrimp appeared to be the most close in its primary structure to that of the king crab (70% identity).
通过在DEAE-葡聚糖凝胶上连续进行离子交换色谱、在经鲑鱼胰蛋白酶水解产物的肽配体修饰的琼脂糖上进行亲和色谱以及在Mono Q柱上进行离子交换色谱，从堪察加拟石蟹的肝胰腺中分离出了均一状态的胰蛋白酶。蛋白质的总产率为64%。测定了其N端氨基酸序列（IVGGTEVTPG-）。获得了帝王蟹肝胰腺的扩增总cDNA样本。基于已知的成熟形式胰蛋白酶的N端氨基酸序列，分离出了包含该基因完整编码部分的cDNA片段。酶原的多肽链由266个氨基酸组成。成熟胰蛋白酶包含237个氨基酸，这与其24.8 kDa的分子量相对应。将帝王蟹胰蛋白酶的氨基酸序列与其他甲壳类动物胰蛋白酶的氨基酸序列进行比较，显示出它们具有高度的结构同源性。南美白对虾的胰蛋白酶在一级结构上似乎与帝王蟹的最为接近（同一性为70%）。
