带有突变穿膜肽重组凋亡素可溶性表达的研究

目的构建apoptin（凋亡素）-HBDCK-pET28a突变体重组表达载体,在大肠杆菌BL21（DE3）中实现高效可溶性表达,并观察突变后穿膜肽（CPP）在HeLa细胞中的转导活性。方法采用PCR介导突变方法将重组蛋白apoptin-HBD及EGFP-HBD的HBD结构域中的Cys（C）突变为Lys（K）。突变的重组质粒在大肠杆菌BL21（DE3）中表达,Ni2＋-NTA亲和层析纯化目的蛋白,进一步观察HeLa细胞突变后HBD的转导活性。结果经双酶切鉴定和序列分析,突变后的重组质粒apoptin-HBDCK-pET28a及EGFP-HBDCK-pET28a构建正确。转化E.coli BL21（DE3）后,融合蛋白均获得可溶性表达。EGFP-HBDCK作用于HeLa细胞13 h后,可观察到细胞内强绿色荧光。结论 HBD结构域中C突变为K,可达到融合蛋白可溶性表达的目的,且仍能保持很强的转导活性,可携带EGFP蛋白进入HeLa细胞。

Soluble Expression of Recombinant Apoptin Fused with Mutant Cell-penetrating Peptide

Objective A recombinant expression vector of mutant apoptin-HBDCK-pET28a was constructed to investigate whether the recombinant proteins were highly expressed in a soluble form in E.coli BL21（DE3） and tested the mutant cell-penetrating peptide（CPP） transduction activity in HeLa cells.Methods A PCR mutation approach was used for the expressions of mutated apoptin-HBDCKand EGFP-HBDCK,which Cys（C） in heparin-binding domain（HBD） was mutated into Lys（K）,respectively.The mutated fusion proteins were expressed in E.coli BL21（DE3） and purified by Ni2＋-NTA affinity chromatography.The transduction activity of the mutant CPP in HeLa cells was detected.Results Both restriction enzyme and sequencing analysis proved that the mutant recombinant plasmid apoptin-HBDCK-pET28a and EGFP-HBDCK-pET28a were constructed correctly.Both fusion proteins were expressed in a soluble form in E.coli BL21（DE3）.After a 13h incubation of HeLa cell with EGFP-HBDCK,an intensive green fluorescence was observed in most cells.Conclusion The fusion proteins can be expressed in a soluble form after a CK mutation was made in HBD domain.The mutant CPP possesses a good transduction activity and can bring fused EGFP protein into HeLa cells.