Srinivasan Sriram, Barnard Gavin C, Gerngross Tillman U
Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, New Hampshire 03755, USA.
Biotechnol Bioeng. 2003 Oct 5;84(1):114-20. doi: 10.1002/bit.10756.
We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli.
我们之前报道了一种基于真养产碱菌的新型蛋白质表达系统的开发。在本研究中，我们报告了基因拷贝数对真养产碱菌中重组蛋白表达的影响。我们比较了基于质粒的表达系统与染色体整合的重组基因稳定性和表达水平。编码有机磷水解酶（OPH）的基因的单拷贝、双拷贝和三拷贝被整合到真养产碱菌染色体中，该酶在大肠杆菌中易于形成包涵体。发现可溶性活性OPH浓度与基因拷贝数之间呈线性增加。使用三拷贝整合体，我们能够在高细胞密度发酵中产生约4.3 g/L的OPH。这代表了迄今为止该酶报道的最高滴度，约为大肠杆菌中报道的表达水平的30倍。
