Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks.
Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks.
复制标题
DOI：
10.1186/s12934-014-0127-y
复制
发表时间：
2014-09-04
影响因子：
6.4
通讯作者：
Bill RM
中科院分区：
文献类型：
--
作者：
Bawa Z;Routledge SJ;Jamshad M;Clare M;Sarkar D;Dickerson I;Ganzlin M;Poyner DR;Bill RM
巴斯德毕赤酵母是一种广泛用于重组蛋白生产的宿主;表达通常由甲醇诱导的醇氧化酶（AOX）启动子驱动。最近，该系统已成为结构生物学和药物发现的重组G蛋白偶联受体（GPCR）的重要来源。已经研究了不同培养参数（例如pH、溶解氧浓度、培养基组成、消泡剂浓度和培养温度）对巴斯德毕赤酵母中广泛的重组蛋白的生产率的影响。相比之下，尚未仔细研究预诱导阶段对产率的影响。在本研究中，我们检查了产生三种不同重组蛋白的巴斯德毕赤酵母生物反应器培养的预诱导阶段：GPCR、人A2 a腺苷受体（hA 2aR）、绿色荧光蛋白（GFP）和人降钙素基因相关肽受体组分蛋白（作为GFP融合蛋白; hCGRP-RCP-GFP）。在甘油生长的巴斯德毕赤酵母的IL生物反应器培养的预诱导阶段中检测到功能性hA 2aR。在单独的实验中，甘油生长的巴斯德毕赤酵母菌株在甲醇添加之前分泌可溶性GFP。当葡萄糖，这已被证明抑制AOX表达，是预诱导碳源，hA 2aR和GFP仍然产生在预诱导阶段。hA 2aR和GFP也在无甲醇培养中产生;在碳源耗尽后，功能性蛋白质产量保持或增加。对10 L中试规模培养的诱导前阶段的分析也表明，即使在存在细胞毒性浓度的甲醇的情况下，甲醇诱导后至少保持了诱导前产率。hCGRP-RCP-GFP的额外生物反应器数据和GFP、辣根过氧化物酶（HRP）、人四跨膜蛋白hCD 81和CD 82以及紧密连接蛋白人密蛋白-1的摇瓶数据证明，生物反应器而非摇瓶培养在巴斯德毕赤酵母培养物的预诱导阶段中表现出重组蛋白生产。重组hA 2aR、GFP和hCGRP-RCP-GFP的产生可以在甲醇诱导之前在生物反应器培养中检测到，而GFP、HRP、hCD 81、hCD 82和人密蛋白-1的摇瓶培养不是这种情况。这证实了早期的建议，从AOX启动子，我们在这里报告甘油和葡萄糖生长的细胞在生物反应器培养泄漏表达。这些研究结果表明，AOX依赖的生物过程的生产力并不仅仅依赖于甲醇的诱导。我们的结论是，为了最大限度地提高总产量，预诱导阶段的培养条件应进行优化，提高比生产率可能会导致生物量产量下降。本文的在线版本（doi：10.1186/s12934-014-0127-y）包含补充材料，可供授权用户使用。
Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake-flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields. The online version of this article (doi:10.1186/s12934-014-0127-y) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
3.7
作者：
Bonander N;Jamshad M;Oberthür D;Clare M;Barwell J;Hu K;Farquhar MJ;Stamataki Z;Harris HJ;Dierks K;Dafforn TR;Betzel C;McKeating JA;Bill RM
通讯作者：
Bill RM
影响因子：
64.8
作者：
Hino, Tomoya;Arakawa, Takatoshi;Iwanari, Hiroko;Yurugi-Kobayashi, Takami;Ikeda-Suno, Chiyo;Nakada-Nakura, Yoshiko;Kusano-Arai, Osamu;Weyand, Simone;Shimamura, Tatsuro;Nomura, Norimichi;Cameron, Alexander D.;Kobayashi, Takuya;Hamakubo, Takao;Iwata, So;Murata, Takeshi
通讯作者：
Murata, Takeshi
影响因子：
2.8
作者：
Inan, M;Meagher, MM
通讯作者：
Meagher, MM
影响因子：
3.8
作者：
Cunha, AE;Clemente, JJ;Carrondo, MJT
通讯作者：
Carrondo, MJT
影响因子：
46.9
作者：
De Schutter, Kristof;Lin, Yao-Cheng;Callewaert, Nico
通讯作者：
Callewaert, Nico