Center for Agricultural Biotechnology and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
Biotechnol Bioeng. 1993 Mar 5;41(5):557-65. doi: 10.1002/bit.260410508.
In this work, effective feeding schemes that would minimize stress responses to cloned-protein overexpression are investigated. The cloned-protein (chloramphenicolacetyl-transferase, CAT) contains a high aromatic amino acid content, most notably a high phenylalanine content. Experiments performed on Escherichia coli RR1 [pBR329] (constitutive promoter) and E. coli JM105 [pSH101] (inducible promoter) demonstrated that phenylalanine addition increases the rate of synthesis and yield of CAT. A previous study correlating inducer strength with CAT expression in E. coli JM105 [pSH101] indicated that the highest expression rate was accompanied by the highest apparent rate of protein degradation. In this work, the combined addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) and phenylalanine at intermediate levels resulted in substantial increase of CAT synthesis and partial reduction of protein degradation. Furthermore, transmission electron micrographs verified the absence of inclusion bodies, which, along with proteases, were suspected to reduce protein activity. The research demonstrates that significant enhancement in production and stability of heterologous proteins is possible by designing feeding strategies that incorporate knowledge of the interaction between primary cellular metabolism and foreign protein expression.
在这项工作中，我们研究了能够将克隆蛋白过表达引起的应激反应最小化的有效补料方案。该克隆蛋白（氯霉素乙酰转移酶，CAT）含有高芳香族氨基酸含量，尤其是高苯丙氨酸含量。在大肠杆菌 RR1[pBR329]（组成型启动子）和大肠杆菌 JM105[pSH101]（诱导型启动子）上进行的实验表明，添加苯丙氨酸可以提高 CAT 的合成速度和产量。先前的研究表明，诱导剂强度与大肠杆菌 JM105[pSH101]中 CAT 表达的相关性表明，最高表达率伴随着最高的蛋白质降解表观速率。在这项工作中，在中间水平上同时添加异丙基-β-D-硫代半乳糖苷（IPTG）和苯丙氨酸，导致 CAT 合成的大量增加和蛋白质降解的部分减少。此外，透射电子显微镜证实了不存在包含体，包含体和蛋白酶都被怀疑会降低蛋白质活性。该研究表明，通过设计补料策略，将初级细胞代谢与外源蛋白表达之间的相互作用的知识结合起来，可以显著提高异源蛋白的产量和稳定性。
