利用毕赤酵母系统表达重组人生长激素的研究

Expression and Purification of Recombinant Human Growth Hormone in Pichia pastoris

为了获得重组人生长激素在毕赤酵母中高表达的菌株,按毕赤酵母基因密码子偏爱性,人工合成hGH的全基因序列,该基因被克隆到穿梭质粒pPIC9K中,PEG1000介导转入毕赤酵母GS115细胞,通过G418筛选获得高拷贝转化子.在甲醇的诱导下,实现了hGH在毕赤酵母中的成功表达.通过发酵条件的优化,发酵上清中的表达量达1537mg/L.经过超滤和两步层析,重组蛋白的得率达35%,纯度为97%,相对分子质量测定表明重组蛋白的相对分子质量与理论值相近,N-端氨基酸测序证实hGH基因在毕赤酵母中获得正确的表达.

In order to express the hGH in Pichia pastoris, total gene of human growth hormone was synthesized according to partial gene code of yeast （Pichia pastoris ）. The gene fragment was inserted into shuttle plasmid pPIC9K, then the recombinant plasmid ppIC9K-hGH was transformed into Pichia pastoris strains GS115 with PEG1000 and screening the muhicopy clones by G418 in YPD plates. In the inducement of methanol, recombinant hGH was successfully expressed in Pichia pastoris. After optimizing the fermentation conditions, the yield of recombinant protein reached 1 537 mg/L in the supernatants, the purity of recombinant hGH can reach 97% after one step uhrafihration and two step purification, and the 35% of the total protein can be obtained after optimizing the purication method. Mass spectrometry analyses showed the mass of the rhGH was close to the theoretic value. Amino acid sequencing of N-terminal indicated the rhGH was expressed successfully.