Sinha Jayanta, Plantz Bradley A, Zhang Wenhui, Gouthro Mark, Schlegel Vicki, Liu Chih-Ping, Meagher Michael M
Biological Process Development Facility, Department of Chemical Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68583, USA.
Biotechnol Prog. 2003 May-Jun;19(3):794-802. doi: 10.1021/bp025744q.
Recombinant ovine interferon-tau (r-oIFN-tau) production by Pichia pastoris was studied using methanol as the sole carbon source during induction. The cells were grown on glycerol up to a certain cell density before induction of the AOX1 promoter by methanol for expression of the recombinant protein. Cell growth on methanol has been modeled using a substrate-feed equation, which served as the basis for an effective computer control of the process. The r-oIFN-tau concentration in the culture began to decline despite continued cell growth after 50 (+/- 6) h of induction, which was associated with an increase in proteolytic activity of the fermentation broth. A specific growth rate of 0.025 h(-1) was found to be optimal for r-oIFN-tau production. No significant improvement in r-oIFN-tau production was observed when the specific growth rate was stepped up before the critical point when r-oIFN-tau concentration started decreasing during fermentation. However, best results were obtained when the specific growth rate was stepped down from 0.025 to 0.02 h(-1) at 38 h of induction, whereby the active production period was prolonged until 70 h of induction and the broth protease activity was correspondingly reduced. The corresponding maximum protein yield was 391.7 mg x L(-1) after 70 h of fermentation. The proteolytic activity could be reduced by performing fermentations at specific growth rates of 0.025 h(-1) or below. The recombinant protein production can be performed at an optimal yield by directly controlling the methanol feed rate by a computer-controlled model. The production profile of r-oIFN-tau was found to be significantly different from other secreted and intracellular recombinant protein processes, which is an indication that recombinant protein production in Pichia pastoris needs to be optimized as individual processes following established principles.
研究了在诱导过程中以甲醇作为唯一碳源，利用毕赤酵母生产重组羊干扰素 -τ（r - oIFN -τ）的情况。细胞先在甘油上生长至一定细胞密度，然后通过甲醇诱导AOX1启动子以表达重组蛋白。甲醇上的细胞生长已用底物进料方程进行建模，该方程为该过程的有效计算机控制提供了基础。诱导50（±6）小时后，尽管细胞持续生长，但培养物中r - oIFN -τ浓度开始下降，这与发酵液中蛋白水解活性的增加有关。发现0.025 h⁻¹的比生长速率最适合r - oIFN -τ的生产。在发酵过程中r - oIFN -τ浓度开始下降的临界点之前提高比生长速率时，未观察到r - oIFN -τ产量有显著提高。然而，在诱导38小时时将比生长速率从0.025降至0.02 h⁻¹可获得最佳结果，此时活性生产期延长至诱导70小时，且发酵液蛋白酶活性相应降低。发酵70小时后相应的最大蛋白产量为391.7 mg·L⁻¹。通过以0.025 h⁻¹或更低的比生长速率进行发酵可降低蛋白水解活性。通过计算机控制模型直接控制甲醇进料速率，可实现重组蛋白的最优产量生产。发现r - oIFN -τ的生产曲线与其他分泌型和细胞内重组蛋白生产过程有显著不同，这表明在毕赤酵母中生产重组蛋白需要按照既定原则作为单独过程进行优化。
