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蛋白质内含子介导的断裂(切割)反应被用于蛋白质纯化、连接和环化等,但目前仍存在断裂效率低、断裂反应的不可控、产物复杂等问题。蛋白质内含子的定点突变可导致其N端或C端断裂。其末位氨基酸突变则剪接反应第3步天冬酰胺环化无法进行,发生N端断裂;其首位氨基酸发生突变则剪接反应第一步酰基重排及其后续步骤均无法进行,而天冬酰胺环化仍可进行,发生C端断裂。利用已获得的高剪接活性的S1和S11型断裂蛋白质内含子Ssp GyrB,分别将其参与剪接反应的首位半胱氨酸或末位天冬酰胺突变为丙氨酸,构建能够发生一端断裂的断裂蛋白质内含子。研究结果表明,突变后断裂蛋白质内含子的剪接反应几乎不发生,其断裂活性有不同程度的提高,获得了在大肠杆菌体内具有较高效断裂活性的断裂蛋白质内含子。这将为进一步研究其体外可控性剪接、构建高效的蛋白纯化系统和深入研究蛋白质内含子的剪接机制提供基础。
Protein intron-mediated cleavage (cleavage) reaction is used for protein purification, ligation and cyclization, but there are still some problems such as low efficiency of cleavage, uncontrollable cleavage reaction and complex products. Site-directed mutagenesis of a protein intron causes its N-terminal or C-terminal cleavage. The last amino acid mutation splicing reaction asparaginase step 3 can not be carried out, N-terminal cleavage occurs; the first amino acid mutation occurs then the first step of the splicing reaction acyl rearrangement and subsequent steps are not, and asparagine Cyclization can still proceed with C-terminal cleavage. Using the obtained high-splicing activities of S1 and S11 fragmented intron Ssp GyrB, the first cysteine or the last asparagine participating in the splicing reaction were respectively mutated to alanine to construct a protein capable of splitting at one end Break the protein intron. The results showed that the splicing reaction of the split protein intron after mutation hardly occurred, and the activity of its cleavage increased to some extent, and the cleaved protein intron with more efficient cleavage activity in E. coli was obtained. This will provide a basis for further study of its in vitro controllable splicing, construction of a highly efficient protein purification system and further study of the splicing mechanism of the protein intron.