Shin C S, Hong M S, Bae C S, Lee J
Bioprocess Engineering Laboratory, Hanhyo Institutes of Technology, Yusong-Ku, Taejon, South Korea.
Biotechnol Prog. 1997 May-Jun;13(3):249-57. doi: 10.1021/bp970018m.
Synthesis of recombinant protein (human mini-proinsulin) is investigated in fed-batch cultures at high cell concentration of recombinant Escherichia coli BL21(DE3)[pET-3aT2M2]. Transcription of the recombinant gene is controlled by a T7 promoter system. The human mini-proinsulin is characterized by a C-chain peptide consisting of only nine amino acids, whereas the C-chain peptide of natural human proinsulin is made up of 35 amino acids. It is expressed in a fusion protein with a small fusion partner (a peptide with 18 amino acids) and finally aggregated into insoluble inclusion bodies in cytoplasm of recombinant E. coli. The fermentative production of this small fusion mini-proinsulin may be of great advantage in enhancing the yield of human insulin. To find an optimum induction strategy, effects of various key cultivation variables on the mini-proinsulin production are examined in high cell density fed-batch cultures. No general correlation is found between preinduction specific growth rate and recombinant protein synthesis, which confers a flexibility in choosing the feeding strategy of preinduction media for achieving the high cell density cultures. A culture temperature below 37 degrees C is unfavorable for recombinant gene expression, and the T7-based expression system is almost completely repressed at 30 degrees C. The nutrient glucose and yeast extract concentration in postinduction feed media is optimized by applying a statistical method for medium optimization, i.e. response surface methodology, and an effective amount of inducer molecule (IPTG) is determined to maximize the specific recombinant protein formation. The mini-proinsulin production in E. coli culture is significantly influenced by the volumetric feed rate of postinduction media, which is shown to be closely related to the plasmid copy number in the recombinant cell. Consequently, in a single-stage fed-batch process, the mini-proinsulin concentration is increased up to 7 g/L, approximately 62 wt % of which corresponds to mature human insulin. A two-stage fed-batch fermentation process, with recombinant cell growth occurring at a constant growth rate and constant cell concentration in a growth fermenter and mini-proinsulin production in an induction fermenter, is designed, and its efficacy in increasing volumetric productivity of mini-proinsulin is demonstrated.
在重组大肠杆菌BL21(DE3)[pET-3aT2M2]的高细胞浓度补料分批培养中研究了重组蛋白（人迷你胰岛素原）的合成。重组基因的转录由T7启动子系统控制。人迷你胰岛素原的特征在于其C链肽仅由九个氨基酸组成，而天然人胰岛素原的C链肽由35个氨基酸组成。它以与小融合伴侣（一种含18个氨基酸的肽）的融合蛋白形式表达，最终在重组大肠杆菌的细胞质中聚集成不溶性包涵体。这种小融合迷你胰岛素原的发酵生产在提高人胰岛素产量方面可能具有很大优势。为了找到最佳诱导策略，在高细胞密度补料分批培养中研究了各种关键培养变量对迷你胰岛素原生产的影响。诱导前比生长速率与重组蛋白合成之间未发现普遍相关性，这为选择诱导前培养基的补料策略以实现高细胞密度培养提供了灵活性。低于37℃的培养温度不利于重组基因表达，基于T7的表达系统在30℃时几乎完全被抑制。通过应用统计方法进行培养基优化，即响应面法，优化诱导后补料培养基中的营养葡萄糖和酵母提取物浓度，并确定有效量的诱导分子（IPTG）以最大化重组蛋白的比形成量。大肠杆菌培养物中迷你胰岛素原的生产受到诱导后培养基体积补料速率的显著影响，这表明其与重组细胞中的质粒拷贝数密切相关。因此，在单阶段补料分批过程中，迷你胰岛素原浓度提高到7 g/L，其中约62 wt%对应于成熟人胰岛素。设计了一种两阶段补料分批发酵过程，在生长发酵罐中重组细胞以恒定生长速率和恒定细胞浓度生长，在诱导发酵罐中生产迷你胰岛素原，并证明了其在提高迷你胰岛素原体积生产率方面的有效性。
