人卵透明带蛋白3差异表达菌的鉴定及比较

Identification and comparison of the variety expression of recombinant human zona pellucida-3

目的:研究重组人卵透明带ZP3蛋白的差异表达情况。方法:构建8个重组rhZP3的差异表达质粒在毕赤酵母中进行诱导表达,研究跨膜区保留与否、潜在酶切位点突变与否及多聚组氨酸纯化标签设计的位置是否影响rhZP3的表达情况。结果:保留跨膜区会影响重组蛋白泌出胞外而使表达量降低,潜在酶切位点存在会导致重组蛋白被不完全降解,而多聚组氨酸纯化标签设计位置对表达也有一定影响。结论:从表达量和产物完整性综合考虑,以突变型成熟肽为佳,含跨膜区的蛋白则应选用pPICZaA质粒,以便富集得到完整的表达蛋白。

Aim：To investigate the variety of the recombinant human zona pellucida-3 （rhZP3） expression in Pichia pastoris. Methods： Eight recombinants were constructed in order to study whether the expression of rhZP3 will be influenced by the following factors, which include retaining of the transmembrane domain of rhZP3, mutation of the potential sites of protease, and the position of His-tag. Results： The presence of the transmembrane domain decreased expression of rhZP3. The potential protease cleavage sites induced the incomplete degradation of rhZP3. And the position of His-tag would affect the expression of rhZP3 in some ways. Conclusion： The mutated protein without transmembrane domain would be the best one considering the yield and integrity. And pPICZaA is well for expression of rhZP3 with transmembrane domain.