大肠埃希菌内锌离子对断裂内含肽Npu DnaE自剪接作用的抑制效应

Inhibitory Effect of Intracellular Zinc Ions on the Splicing of Npu DnaE

内含肽是前体未成熟蛋白质中的一段具有自我剪接功能的序列,其活性受多种因素影响,如锌离子浓度的控制。为了在大肠埃希菌胞内实现天然断裂型内含肽Npu DnaE"自剪接"作用可控,表达了Npu DnaE内含肽C-末端和人Ig G1抗体Fc段融合蛋白,及Npu DnaE内含肽N-末端和铜绿假单胞菌外毒素(PE)截短型融合蛋白,并加入浓度由1 mmol/L增加至2.5 mmol/L的锌离子。结果大肠埃希菌的生长没有受到影响,Fc蛋白和PE蛋白的单独表达没有受影响,而内含肽N-末端和C-末端发生剪接作用后,外显肽Fc和PE融合蛋白的表达量显著降低。上述结果表明,在大肠埃希菌表达富含半胱氨酸目的蛋白时添加锌离子,可以有效地抑制胞内Npu DnaE内含肽的"自剪接"作用,为拓展内含肽在生物药物生产中的应用提供了试验依据。

Intein is a part of polypeptide in the premature protein with the capability of self-splicing, whose activity can be affected by many factors, including zinc ions. It has been proved that zinc ions can inhibit the cleavage reaction of inteins in vitro by binding to the sulfhydryl. In order to inhibit the self-splicing of Npu DnaE intein in Escherichia coli cells during protein expression, the DnaE N-terminal Fc fusion protein and DnaE C-terminal PE fusion protein were co-expressed in E.coli cells in the medium supplemented with zinc ions. The growth ofE. coli cells as well as the expression of Fc and PE proteins were unaffected with the increasing of zinc ion concentration from 1 mmol/L to 2.5 mmol/L, but the formation of the spliced product Fc-PE fusion proteins significantly decreased. These results showed that addition of zinc ions could effectively inhibit the intracellular Npu DnaE intein splicing, which realized the inhibition in the cells of E. coli for the first time. The result further expands the application of the inteins in the host cells, and provides an experimental basis for the application of intein in the process of biological drug production.