Sung W L, Zahab D M, Barbier J R, Watson D, Yaguchi M, Neugebauer W, Willick G E
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
J Biol Chem. 1991 Feb 15;266(5):2831-5.
Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.
合成的人甲状旁腺激素（PTH）基因氨基末端区域的特定简并密码子，对这种84个氨基酸的多肽在大肠杆菌中的表达产物和产量都产生了显著影响。用富含腺嘌呤的简并密码子构成PTH-（1-5）区域时，完整的PTH已作为唯一的PTH产物以6.5毫克/升的浓度表达。相比之下，用富含鸟嘌呤的简并密码子，主要产物是类似物PTH-（8-84）。使用富含胞嘧啶或胸腺嘧啶的简并密码子仅产生少量免疫反应性产物（0.2毫克/升）。当氨基末端区域用富含腺嘌呤的简并密码子重建后，合成基因的中部和羧基区域也进行了重建，以模仿大肠杆菌偏好的密码子简并性。表达产生了浓度为20毫克/升的完整PTH。用针对PTH氨基或羧基末端的特异性抗体进行的凝胶电泳和蛋白质印迹分析表明，只有一种与PTH相关的多肽，其迁移率与合成的完整PTH样品相同。氨基酸测序、组成分析、质谱分析以及腺苷酸环化酶生物测定证实，纯化产物为加工后的完整PTH。
