Engineering of recombinant Chinese hamster ovary cells using proteomic investigation = 프로테오믹스 연구를 통한 재조합 CHO 세포의 개량

Abstract

In the culture of recombinant Chinese hamster ovary (rCHO) cells, optimization of culture environmental parameters, such as low temperature, hyperosmolarity, culture pH, and sodium butyrate addition has been widely studied. Although optimizing culture conditions generally takes less time and labor than genetic engineering, it also has some side-effects including decrease of cell growth, viability, or culture longevity. Accordingly, my investigations to improve cell performance were focused on an alternative method, genetic engineering. Selection of target proteins for genetic engineering was carried out using a proteomic approach which finds the changes in protein expression according to changes of culture conditions. Sodium butyrate (NaBu) is known to enhance the specific productivity of Chinese hamster ovary cells expressing human thrombopoietin. In order to better understand the intracellular responses of these cells resulting from NaBu treatment, the proteomic profiles of cells treated with various concentrations of NaBu (0-3mM) were compared using two-dimensional electrophoresis (2-DE). Based on spot intensities, 80 high intensity protein spots were selected. Fifty-six of the 80 protein spots, which represent 28 different kinds of proteins, were identified by MALDI-TOF-MS and MS/MS. Compared to control without NaBu treatment, the expression levels of 2 proteins (glucose regulated protein 78 (GRP 78) and peroxiredoxin 4) were increased over two fold with NaBu treatment and the expression level of phosphopyruvate hydratase was decreased over two fold with NaBu treatment. Due to multiplicity (multiple spots for one protein), a change in one single spot intensity from a 2-DE gel image may not represent the total change in expression level for that protein. Western blot analyses of GRP78, HSC70 and ERp57 confirmed the results of the MS analyses, though a degree of change in expression level differed between the two methods. Accordingly, the proteomic approa...