Engineering of Pichia pastoris for improved production of antibody fragments
Engineering of Pichia pastoris for improved production of antibody fragments
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DOI：
10.1002/bit.20851
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发表时间：
2006-06-05
影响因子：
3.8
通讯作者：
Mattanovich, Diethard
中科院分区：
文献类型：
--
作者：
Gasser, Brigitte;Maurer, Michael;Mattanovich, Diethard
甲醇营养型酵母巴斯德毕赤酵母已被用于表达许多蛋白质，包括抗体片段。然而，限制变得明显，特别是当分泌异源二聚体Fab片段时。迄今为止，抗体片段仅在强诱导型醇氧化酶1（AOX 1）启动子的控制下表达，其可通过过度转录来胁迫细胞。在这里，我们研究了两种不同的单克隆抗HIV 1抗体（2F 5和2G 12）与AOX 1和甘油醛-3-磷酸脱氢酶（GAP）启动子的单链和Fab片段的分泌特性。同时，对不同分泌先导物和菌株的影响进行了评价。有趣的是，仅当使用差距启动子和酿酒酵母交配因子α（MF α前导序列）时才实现分泌，而两种巴斯德毕赤酵母菌株之间没有差异。在2175 Fab表达菌株的补料分批发酵期间，观察到Fab重链的细胞内保留，而仅在上清液中检测到完整Fab和单个轻链分子。这导致的结论是蛋白质折叠和异源二聚体组装在ER中的Fab分泌的限速步骤。为了缓解这一限制，S.酿酒酵母蛋白二硫键异构酶（PDI）和未折叠蛋白应答（UPR）转录因子HAC 1在巴斯德毕赤酵母中组成型过表达。虽然HAC 1的过表达导致Fab分泌适度增加1.3倍，但PD 1使Fab水平增加1.9倍。因此，链间二硫键的形成可以被视为Fab组装和随后分泌的主要速率限制因素。(c)2006 Wiley Periodicals，Inc.
The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments.. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor alpha (MF alpha leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2175 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in-the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PD1 enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. (c) 2006 Wiley Periodicals, Inc.