【作者】 关波；

【导师】 李华钟；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 毕赤酵母能够在高效表达外源蛋白的同时，对外源蛋白进行翻译后的正确折叠和加工修饰，已成为目前表达外源蛋白尤其是人源药物蛋白所广泛采用的表达宿主之一。然而随着越来越多外源蛋白在毕赤酵母中分泌表达，发现某些外源蛋白在分泌表达过程中存在胞内聚集而导致分泌表达量较低，宿主蛋白酶对外源蛋白存在不同程度的降解等问题。本研究室前期研究结果也表明，采用相同的载体和宿主表达系统，在各自优化后的培养和表达条件下，多种人血清白蛋白(HSA)融合蛋白在毕赤酵母中的分泌表达量较HSA显著降低，分泌表达过程中HSA融合蛋白降解较严重，对表达产物的后期纯化带来了相当的困难。基于毕赤酵母存在的以上不足，本论文通过基因工程的手段，从以下三个方面对已有HSA融合蛋白的毕赤酵母分泌表达系统进行了改造：1、为避免诱导碳源甲醇对发酵制备HSA融合蛋白可能带来的不利影响，以毕赤酵母GS115作为出发菌株，成功构建筛选获得一株甘油醛3-磷酸脱氢酶基因启动子(PGAP)调控的分泌表达rhIL-2-HSA融合蛋白的菌株。该菌株能在多种单一碳源的培养基中组成型分泌表达rhIL-2-HSA融合蛋白。以葡萄糖为碳源时，rhIL-2-HSA融合蛋白的表达量最高。5L罐发酵实验结果显示，rhIL-2-HSA融合蛋白的PGAP表达菌株，在以葡萄糖为单一碳源的发酵培养基中发酵60h，rhIL-2-HSA融合蛋白的表达量可达约250mg·L-1。由此纯化获得的rhIL-2-HSA融合蛋白的体外活性约为1.040×106IU·mg-1，与甲醇诱导型的PAOX1表达系统获得的rhIL-2-HSA融合蛋白的体外活性水平相当。2、为促进HSA融合蛋白的分泌表达，选择折叠酶PDI、Ero1、分子伴侣BiP、以及囊泡转运过程参与调控膜融合的SM蛋白Sec1和Sly1作为分泌辅助因子进行共表达，并分析共表达以上分泌辅助因子对rhIL-2-HSA融合蛋白分泌表达的影响。共表达分泌辅助因子PDI、Ero1、BiP、Sec1和Sly1对分泌表达融合蛋白rhIL-2-HSA菌株的正常生长没有影响，分别使融合蛋白rhIL-2-HSA的分泌表达量提高了120%,130%,90%,150%和90%。Western blot对共表达PDI，Ero1，BiP，Sec1和Sly1的各个共表达菌株细胞内胞质蛋白组分和膜偶联蛋白组分进行半定量发现，胞质蛋白组分和膜偶联蛋白组分中rhIL-2-HSA融合蛋白的含量也显著增加。定量PCR分析发现，共表达菌株中相关分泌辅助因子的基因转录也显著增强。3、为了减少HSA融合蛋白在分泌表达过程中的降解，以毕赤酵母GS115菌株为亲本菌株，分别敲除毕赤酵母各个Yapsin蛋白酶编码基因YPS1，YPS2，YPS3，MKC7和YPS7，构建了毕赤酵母单基因敲除菌株Yps1Δ，Yps2Δ，Yps3Δ，Mkc7Δ，Yps7Δ和多基因敲除菌株GBD2，GBD3，GBD4，GBD5。将融合蛋白HSA-ADAM15的表达载体转入以上构建的单基因敲除菌株，发现敲除单个Yapsin蛋白酶基因YPS1，YPS2，YPS3，MKC7或YPS7，对融合蛋白HSA-ADAM15分泌表达时的降解没有明显改善。在多重Yapsin蛋白酶基因敲除菌株GBD4(yps1Δyps2Δyps3Δ)和GBD5(yps1Δyps2Δyps3Δyps7Δ)中分泌表达HSA-ADAM15融合蛋白，HSA-ADAM15的降解得到部分缓解：GBD4菌株表达的完整目标蛋白含量约为对照GS115菌株中的133%，降解片段d1和d2的含量则分别降低了50%和52%。这表明多个Yapsin蛋白酶缺陷的毕赤酵母菌株能够部分缓解分泌表达过程中融合蛋白HSA-ADAM15的降解。此外，对各个蛋白酶缺陷菌株的生长表型进行分析，发现在获得的蛋白酶缺陷菌株中，Yps7Δ菌株对CalcofluorWhite，刚果红和SDS三种细胞壁胁迫性物质的抗性增强。与GS115菌株相比，Yps7Δ突变体细胞侧生细胞壁(lateral cell wall)部分的几丁质含量降低, β-1,3-葡聚糖含量则显著增加。透射电镜观察也发现Yps7Δ突变体细胞壁中由β-1,3-葡聚糖组成的细胞壁内层结构厚度也显著增加，表明毕赤酵母的Yps7参与细胞壁组分合成的调控。生长表型分析还发现，Yps7Δ菌株对由KCl和NaCl引起的渗透胁迫也表现出显著增强的抗性。渗透胁迫条件下，Yps7Δ菌株自身胞内甘油积累量显著高于GS115菌株的积累量，表明毕赤酵母的Yps7还参与渗透胁迫响应的调控。更多还原

【Abstract】 Due to the potential of producing soluble, correctly folded protein with high yield, theyeast Pichia pastoris is currently one of the most widely used hosts for the expression ofheterologous proteins, especially for those recombinant human proteins. However, limitationsof expression efficiency and proteolytic degradation are often reported for many differentheterologous proteins. Secretory expression of a series of peptides genetically fused to humanserum albumin (HSA) in P. pastoris was successfully achieved in previous work of mylaboratory. The secretion levels of different HSA fusion proteins in P. pastoris were muchlower compared with HSA when using the same strain and expression vector under theoptimal cultivation condition. In addition, different HSA fusion proteins usually underwentproteolytic degradation in different degree during secretory expression. To circumvent thesedefects and improve the secretory expression of HSA fusion proteins in P. pastoris, weinvestigated the possible effects of genetic engineering the Pichia expression system of HSAfusion proteins by following three aspects:(1) To avoid the possible negative effect of methanol on the expressed HSA fusion protein,a constitutive expression vector for secretory expression of rhIL-2-HSA fusion protein in P.pastoris was constructed. The coding gene was placed in frame with the Saccharomycescerevisiae α-factor secretion signal sequence under control of the GAP promoter. Theresulting recombinant plasmid pGAPzαA-rhIL-2-HSA was integrated into the genome of theP. pastoris GS115by electroporation. The effect of different carbon sources on secretoryexpression of rhIL-2-HSA fusion protein was evaluated in shaking flask cultures. We foundthat recombinant P. pastoris grew well and rhIL-2-HSA fusion protein was the mostefficiently secreted into the medium when using glucose as carbon source. Fed-batchfermentation strategy using glucose as carbon source for constitutive expression ofrhIL-2-HSA fusion protein was investigated in5-L bioreactor. The expression level ofrhIL-2-HSA could reach about250mg·L-1after60h fermentation. The rhIL-2-HSA fusionprotein produced by this constitutive expression system was purified and exhibited a specificbioactivity of1.040×106IU·mg-1in vitro, which is similar to the rhIL-2-HSA produced byAOX1derived expression system.(2) Several steps in the secretory pathway, such as folding within the ER and vesicletrafficking are suggested to be bottlenecks during recombinant protein secretion in yeast.Three ER resident proteins including immunoglobulin binding protein(BiP), protein disulfideisomerase (PDI) and ER oxidoreductin (Ero1), and two putative Sec1/Munc18(SM) proteins(Sec1and Sly1) involved in membrane fusion during vesicle trafficking were cloned from P.pastoris and selected as potential secretion helper factors to investigate the effects of theiroverexpression on secretory expression of HSA fusion protein rhIL-2-HSA. Constitutiveoverexpression of the five selected secretion factors did not have obvious negative effect oncell growth of the rhIL-2-HSA secreting strain. Among the five secretion helper factors,individually co-overexpression of the Ero1, PDI, BiP, Sec1, and Sly1improved the secretionlevel of rhIL-2-HSA by about120%,130%,90%,150%, and90%comparing to the controlstrain. Western blot analysis of the intracellular level of IL-2-HSA protein showed that thecontent of IL-2-HSA protein in the cytosolic protein fraction and membrane-associated protein fraction in each of the five co-overexpression strains were also increased comparing tothat in the control strain. qPCR analysis also showed that the transcription levels of someother secretion helper factor genes were increased when one secretion helper factor wasoverexpressed.(3) To alleviate the degradation of HSA fusion protein during secretory expression in P.pastoris, the five Yapsin protease genes assigned as YPS1, YPS2, YPS3, YPS7and MKC7in P.pastoris were disrupted individually or simultaneously. We then evaluated the expression of aHSA fusion protein (HSA-ADAM15) in various P. pastoris protease disruption strains (yps1Δ,yps2Δ, yps3Δ, yps7Δ and mkc7Δ). Unfortunately, the degradation of this HSA fusion protein(HSA-ADAM15) during secretory expression was not alleviated in thesesingle-protease-deficient strains. We further constructed several multiple-protease-deficientstrains and used them as hosts for expression of HSA-ADAM15fusion protein in shake-flaskcultivation. SDS-PAGE analysis of the HSA fusion protein (HSA-ADAM15) expressed bythese multiple-protease-deficient strains showed that double disruptants (yps1Δyps2Δ andyps1Δyps3Δ) did not reduce the amount of degradation bands d1(~66kDa) and d2(~45kDa)comparing to the parental strain GS115. The amount of intact HSA-ADAM15in GBD4(yps1Δyps2Δyps3Δ) was about133%of that in strain GS115, and the amount of degradationbands d1and d2were reduced by about50%and52%, respectively, comparing to that instrain GS115. These results showed that the multiple disruptants of Yapsin protease genes arepotential industrially valuable hosts for secretory expression of HSA-ADAM15fusion protein.In addition, among these putative GPI-linked aspartyl proteases, we unexpectly found thatdisruption of PpYPS7gene conferred the yps7Δ mutant cell increased resistance to cell wallperturbing reagents Congo red (CR), Calcofluor white (CW) and SDS. Quantitative analysisof cell wall components showed lower content of chitin and increased amounts ofβ-1,3-glucan. Further staining the cell with Calcofluor white demonstrated that disruption ofPpYPS7gene caused a reduction of the chitin content in lateral cell wall. Consistently, TEMshowed that the inner layer of mutant cell wall, mainly composed of chitin and β-1,3-glucan,was much thicker than that in parental strain GS115. Additionally, yps7Δ mutant alsoexhibited increased resistance to KCl and NaCl compared with parental strain GS115. Thiscould be due to the dramatically elevated intracellular glycerol level in yps7Δ mutant. Theseresults suggested that PpYPS7’s function is involved in cell wall integrity and response toosmotic stress.更多还原