Maywald F, Böldicke T, Gross G, Frank R, Blöcker H, Meyerhans A, Schwellnus K, Ebbers J, Bruns W, Reinhardt G
Department of Genetics, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, F.R.G.
Gene. 1988 Sep 7;68(2):357-69. doi: 10.1016/0378-1119(88)90038-8.
As a basis for a protein design project, we decided to produce the human pancreatic secretory trypsin inhibitor (PSTI) in its active form. Total gene synthesis was carried out efficiently by (i) computer design of the gene fragments, (ii) synthesis of the oligodeoxynucleotides by the segmental support method, and (iii) assembly of double strands under optimized ligation conditions. Fusion to the ompA gene signal peptide led to secretion of processed PSTI in various constructions, with or without additional amino acids (aa) at the N-terminus. The secreted proteins (56 to 63 aa) were biologically active, suggesting that the three cysteine bridges were correctly formed. Surprisingly, after induction the product was found almost exclusively in the culture medium. Variants of PSTI with Asp or Asn at aa positions 21 and 29 [sequences published by Greene et al., Methods Enzymol. (1976) 813-825, and by Yamamoto et al., Biochem. Biophys. Res. Commun. (1985) 605-612] showed the same Ki for both human and porcine trypsin.
作为一个蛋白质设计项目的基础，我们决定以活性形式生产人胰腺分泌型胰蛋白酶抑制剂（PSTI）。通过以下步骤高效地进行了全基因合成：（i）基因片段的计算机设计，（ii）采用分段支持法合成寡脱氧核苷酸，以及（iii）在优化的连接条件下组装双链。与ompA基因信号肽融合导致在各种构建体中分泌加工后的PSTI，N端有无额外氨基酸（aa）。分泌的蛋白质（56至63个氨基酸）具有生物活性，表明三个半胱氨酸桥正确形成。令人惊讶的是，诱导后发现产物几乎完全存在于培养基中。在第21和29位氨基酸处具有天冬氨酸或天冬酰胺的PSTI变体[Greene等人发表的序列，《酶学方法》（1976年）813 - 825，以及Yamamoto等人发表的序列，《生物化学与生物物理研究通讯》（1985年）605 - 612]对人胰蛋白酶和猪胰蛋白酶显示出相同的抑制常数（Ki）。
