Starokadomskiĭ P L, Dubeĭ I Ia, Okunev O V, Irodov D M
Tsitol Genet. 2007 Mar-Apr;41(2):3-11.
Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.
蛋白质剪接是一种翻译后自催化过程，该过程导致从蛋白质前体中切除内部肽段（内含肽），并使侧翼蛋白质序列（外显肽）连接。内含肽介导的蛋白质前体切除具有高度特异性，这使得蛋白质剪接可用于生物技术领域。这项工作旨在在大肠杆菌中获得具有天然N端的人生长激素。构建了由短N端肽、Mxe GyrA内含肽和人生长激素组成的嵌合蛋白。在翻译过程中，甲酰甲硫氨酸修饰的N端肽应通过剪接去除。结果表明内含肽介导了外显肽的切割，但未观察到它们随后的连接。这使得能够制备具有天然N端的人生长激素。研究了各种因素对切割效率的影响。在诱导剂（100 mMβ-巯基乙醇）存在下孵育4 - 6天，嵌合蛋白的切割效率最高（60 - 80%）。
