木质素过氧化物酶LiPH2合成基因在毕赤酵母中的表达

Heterologous expression of a synthetic gene encoding lignin peroxidase H2 in Pichia pastoris

应用化学合成方法获得了碱基序列优化后的木质素过氧化物酶LiPH2合成基因.分别将去除和含有自身信号肽的合成基因与几种选定的表达载体连接,并转化进入相应的Pichiapastoris宿主菌中,共构建了8个不同的毕赤酵母表达系统.对酵母转化子基因及对其发酵液中重组蛋白的分析结果表明,其中1个表达系统的酵母转化子能够成功分泌LiPH2重组蛋白.同时,对影响目的基因表达的各种因素的分析结果表明:就不同宿主菌而言,SMD1168与表达成功的X-33受体菌在其余因素相同的情况下无分泌蛋白表达,pep4基因的缺失对LiPH2蛋白的表达有不利影响;;就不同分泌信号肽而言,与α因子信号肽相比,LiPH2自身信号肽更有利于引导LiPH2的分泌表达;;就不同表达载体而言,其对外源蛋白的表达存在较大差别.本研究中得到的重组蛋白分子量有所增加,说明很可能存在过度糖基化的影响,过度糖基化和C-端氨基酸的增加可能是造成表达蛋白没有活性的原因.

After optimizing the gene sequence, a lignin peroxidase H2 （LiPH2） gene was chemically synthesized and then successlully inserted into selected expression vectors. The recombinant vectors containing different secretion signals and seleetable markers were then transformed into the Pichia pastoris genome. DNA extracted from the recombinant P. pastoris was amplified using the specific primers and analyzed by eleetrophoresis to confirm the correct integration of the LiPH2 gene in the DNA sequence. Eight different recombinant strains were constructed. Only 1 of them successfully expressed LiPH2. Factors that affect the expression were explored and the results are summarized. The host strain SMDl168 was not as effective as X-33 in the expression of the recombinant protein, which indicated that disruption of the pep4 gene in P. pastoris had an adverse effect on the in vitro expression of LiPH2. The native secretion signal of LiPH2 was adequate for both the secretion and expression of the protein. Using vectors pPIC9K and pPIC3.5K led to no expression of LiPH2 in GS115 and SMDl168, but vector pPICZA was more successful. The recombinant protein was in an inactive form. The molecular weight of the recombinant protein was larger than that of the native protein, which suggests that over-glycosylation may be responsible for the inactivation of the enzyme. Tag addition may also influence the protein activation.