【作者】 王海勇；

【导师】 季静；

【作者基本信息】 天津大学 ， 生物分子工程， 2010， 博士

【摘要】 随着后基因组时代的来临,越来越多的蛋白质开发成药物或直接进行分析,这就需要获得合适的宿主来进行异源表达。而为便于后续的纯化过程,分泌性的表达宿主更为理想,虽然在毕赤酵母中获得了较低成本的大量蛋白的异源表达,但因其存在着诱导时间长、受专利保护等因素的限制,国际上针对非常规酵母及丝状真菌的研究逐渐成为热点。UPR信号传导是一个进化上保守的通路,尤其是在酵母和丝状真菌中表现了相当的保守性,由内质网腔的未折叠蛋白累积造成胁迫来自然激活。通路上的转录因子Hac1p在激活、维持这一信号途径中起着关键作用,通过依赖于UPR信号响应元件(UPRE)调节蛋白折叠或降解等相关基因的转录过程,来缓解产生于内质网腔的这种胁迫。为了研究UPR信号的激活对异源表达的影响,我们首先克隆了溶菌酶基因并与α信号肽融合后在酿酒酵母中表达。建立了一个基于平板计数的穿梭质粒拷贝数确定方法,通过质粒的稳定性及拷贝数的研究建立了一步培养的方法使溶菌酶快速表达、分析。本文用逆转录方法克隆了HAC1icDNA,以不同拷贝数的载体或在基因组水平做HAC1的基因去内含子扩增的方式介导UPR信号的持续激活,结果表明UPR信号激活提高异源表达的同时存在着剂量效应。YEplac181介导的UPR激活提高异源表达程度最大,使溶菌酶的表达量跨越了微克级,提高到2.7 mg/L。本文建立了一个新的高通量异源表达菌株筛选系统,本系统不依赖于流式细胞术及微孔板检测仪。依据色谱原理,展示在细胞表面的CBD或亮氨酸拉链与纤维素基质的相互作用成为抵抗来至流动相的“筛选压力”。基于这个系统,通过定向进化技术获得了一个Hac1p突变子(Gly48,Gly111),记为Hac1pm1,异源表达量达到了3.9 mg HEL/L。通过对比Hac1p m1与野生型结构分析推测,氨基酸突变,由于空间位阻减小,可能导致其折叠发生改变,影响其功能。更为重要的是Arg48Gly不但打破了一段α螺旋,使肽链趋向改变走向,更使得亮氨酸拉链基序与DNA的作用发生改变,导致突变子调节的靶基因发生变化。同时也导致实质“剂量”与野生型相比降低,进而调节靶基因的转录发生变化,导致异源表达量的提高。最后,我们通过分析UPR信号途径在转录水平及翻译水平调节的靶基因性质,确定了UPR信号激活过程中菌体生长受抑制的主要原因是氧还平衡的破坏。为进一步提高异源表达量,通过UPRE介导的UPR信号扩增遗传操作,利用KAR2启动子替换MAE1启动子并扩增于ura3基因座,以及敲除基因GPD2来维持氧还平衡。最终,与未进行能荷及氧还平衡调节的对照菌株相比,前者异源表达量是原来的1.4倍(4.9 mg HEL/L),后者为1.2倍(4.4 mg HEL/L)。本文针对UPR这一保守的信号途径在酿酒酵母中进行了系列遗传操作,期望在其他微生物中得到应用。更多还原

【Abstract】 Many natural proteins were developed into drugs and produced for direct analysis, requiring improved hosts to achieve high-level heterologous proteins production. The unfolded protein response (UPR) is an evolutionarily conserved mechanism (especially the yeast and filamentous fungi) by which all eukaryotic cells adapt to the accumulation of unfolded proteins in endoplasmic reticulum (ER). Hac1p, the transcription factor can activate the UPR signal transduction, which regulates genes related the protein folding, degradation and etc.To investigate the effects of engineering UPR pathway to heterologous expression, hen egg white lysozyme (HEL) cDNA cloned from tissue of mature hen oviduct fused toα-signal peptide was inserted into pYES2 and expressed as a modle protein. The shuttle plasmids copy number was assessed using a novel method based on the E.coli transformation and plate count technique, meanwhile, the plasmids stability was estimated. Results proposed one-step cultivation strategy was a feasible alternative.HAC1icDNA was gained by RT-PCR and cloned into different plasmids or substituted the HAC1locus on genome. The results indicated the effect of UPR pathway activation on heterologous expression was dose-dependent, and yeast strain W303 harboring HAC1icDNA on a multi-copy plasmid YEplac181 achieved the highest yields, 2.7 mg HEL/L.A novel high-throughput screening (HTS) system to obtain heterologous over-expression in Saccharomyces cerevisiae strains was developed. The protocol designed here was based on bio-macromolecular physical interaction between CBD or leucine zipper displayed on the surface of hosts and the cellulose substrate. Using the screening system, directed evolution of Hac1p was carried out in Saccharomyces cerevisiae, and an improved mutant (Hac1pm1, Arg48Gly, Ser111Gly, 3.9 mg HEL/L yields) were selected. Results proposed that through selective pressure, this assay may afford a more effective screening system compared with previous selection system. Moreover, it could be employed in general biochemical analysis without utilization of flow cytometry or well plate reader.At last, through UPR pathway analysis, we found that the reduction-oxidation (Redox) balance disruption of strains by UPR activation was the main reason for growth deficit. UPRE (UPR element) mediating the signal augmentation of UPR pathway was performed to improve the production. Truncated form, MAE1s under the control of the promoter of KAR2, harboring UPRE sequence was amplified into the ura3 locus, and 1.4 fold yield was gained compared with the control (W303 +Hac1pm1) when UPR pathway was activated by Hac1pm1. Further, GPD2 was knocked out to balance the Redox and achieved more large heterologous expression.Engineering UPR pathway is expected to improve the heterologous expression in other microorganisms (P.pastoris, K.lactis, filamentous fungi, etc.).更多还原