Wallace C J, Guillemette J G, Hibiya Y, Smith M
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
J Biol Chem. 1991 Nov 15;266(32):21355-7.
If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.
如果定点诱变可用于促进蛋白质半合成，那么就应实现单独使用任何一种技术都无法达成的结构工程目标。我们通过将酵母细胞色素c的Ser65突变为甲硫氨酸来测试这种可能性，从而创建一个用于溴化氰裂解的新位点。发现在此处裂解得到的片段可协同重折叠，使断裂点末端聚集在一起并导致高效的自催化肽键合成。经结构修饰的片段可替代天然片段。通常，由于简要讨论过的原因，天然存在的位点不适用于自催化重新连接，因此这种新方法的优势在于可自由选择适合半合成目标的位点，包括酶切位点。
