Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures.
Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures.
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DOI：
10.1007/s00253-013-5186-1
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发表时间：
2013-11
影响因子：
5
通讯作者：
Oliver, Stephen G.
中科院分区：
文献类型：
--
作者：
Hesketh, Andrew R.;Castrillo, Juan I.;Sawyer, Trevor;Archer, David B.;Oliver, Stephen G.
关键词：
毕赤酵母作为异源蛋白表达的宿主系统在学术界和工业界都得到了广泛的应用。生产通常是通过补料分批诱导过程完成的，已知该过程对细胞生理有负面影响，对蛋白质产量和质量都有限制。我们分析了在趋化培养物中重组蛋白的产生，以了解与甲醇诱导的两种不同程度错误折叠的人溶菌酶变体相关的生理反应。在趋化器的稳态生长过程中，与营养胁迫或生长速率相关的混淆变量被最小化。在非诱导和诱导稳定状态下获得的转录组水平数据的比较发现，在易于细胞内聚集的淀粉样原性人溶菌酶变体（I56T）或错误折叠但可分泌的变体（T70N）的产生过程中，只有约1%的基因组表达发生了变化，表明对它们的产生几乎完全适应。在稳定状态之间的过渡期间，特别是在诱导T70N变异合成之后，观察到一个明显的，但暂时的应激反应，涉及未折叠蛋白反应（UPR）和er相关降解途径，并伴随着大约50个反义转录物的表达变化。结果表明，最佳的异源蛋白产量可以通过连续的过程来实现，该过程可以最大限度地减少培养过程中甲醇诱导的瞬变次数。即使在没有重组蛋白诱导的情况下，也发现在所使用的培养条件下，UPR所需的HAC1 mRNA的加工是组成的。本文的在线版本（doi:10.1007/s00253-013-5186-1）包含补充资料，仅供授权用户使用。
Pichia pastoris is widely used as a host system for heterologous protein expression in both academia and industry. Production is typically accomplished by a fed-batch induction process that is known to have negative impacts on cell physiology that impose limits on both protein yields and quality. We have analysed recombinant protein production in chemostat cultures to understand the physiological responses associated with methanol-induced production of two human lysozyme variants with different degrees of misfolding by P. pastoris. Confounding variables associated with nutrient stress or growth-rate are minimised during steady-state growth in chemostats. Comparison of transcriptome-level data obtained during the non-inducing and inducing steady states identified changes in expression of only about 1 % of the genome during production of either an amyloidogenic human lysozyme variant prone to intracellular aggregation (I56T) or a misfolded but secretable variant (T70N), indicating near-complete acclimation to their production. A marked, but temporary, stress response involving both the unfolded protein response (UPR) and ER-associated degradation pathway was observed during the transient between steady states, particularly following induction of the T70N variant synthesis, and was accompanied by changes in expression of around 50 antisense transcripts. The results suggest that optimal heterologous protein production could best be achieved by a continuous process that minimises the number of methanol-induced transients experienced by the cultures. The processing of HAC1 mRNA required for the UPR was found to be constitutive in the culture conditions used, even in the absence of recombinant protein induction. The online version of this article (doi:10.1007/s00253-013-5186-1) contains supplementary material, which is available to authorized users.
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影响因子：
4.4
作者：
Carvalho ND;Jørgensen TR;Arentshorst M;Nitsche BM;van den Hondel CA;Archer DB;Ram AF
通讯作者：
Ram AF
影响因子：
4.4
作者：
Gasser B;Maurer M;Rautio J;Sauer M;Bhattacharyya A;Saloheimo M;Penttilä M;Mattanovich D
通讯作者：
Mattanovich D
影响因子：
14.9
作者：
Cvijovic, Marija;Olivares-Hernandez, Roberto;Nielsen, Jens
通讯作者：
Nielsen, Jens
影响因子：
4.1
作者：
Jungo, Carmen;Marison, Ian;von Stockar, Urs
通讯作者：
von Stockar, Urs
影响因子：
46.9
作者：
De Schutter, Kristof;Lin, Yao-Cheng;Callewaert, Nico
通讯作者：
Callewaert, Nico