Strandberg L, Enfors S O
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Appl Environ Microbiol. 1991 Jun;57(6):1669-74. doi: 10.1128/aem.57.6.1669-1674.1991.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.
对在大肠杆菌中生产由金黄色葡萄球菌蛋白A和大肠杆菌β-半乳糖苷酶组成的融合蛋白过程中影响包涵体形成的不同参数进行了研究。融合蛋白的胞内表达由pR启动子及其温度敏感阻遏物控制。诱导温度、培养基的pH值以及蛋白A和β-半乳糖苷酶之间连接区氨基酸序列的变化对包涵体的形成有深远影响。在42℃时，包涵体仅在诱导后的最初几个小时形成，此后进一步产生的所有重组蛋白均以可溶且有活性的状态出现。在39℃和44℃下生产导致在整个生产期间形成包涵体，所产生的重组蛋白中有15%至20%以包涵体形式出现。在不控制pH值的情况下培养细胞会导致在整个诱导期形成包涵体，并且包涵体形成随着pH值降低而增加，并且至少部分不溶性蛋白是由细胞内可溶性融合蛋白库形成的。融合蛋白两部分之间连接区氨基酸序列的变化消除了包涵体的形成。
