酵母抽提物促进昆虫细胞生长和重组蛋白生产的特性研究。
Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production.
机构信息
Animal Cell Technology Group and Chemical Biology Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, QC, Canada, H4P 2R2.
出版信息
Cytotechnology. 2007 May;54(1):25-34. doi: 10.1007/s10616-007-9062-7. Epub 2007 Jun 5.
Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1-F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L(-1), fraction F1 showed 71% Sf-9 cell growth improvement and 22% beta-galactosidase production enhancement over YUF (at 1 g L(-1 )in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L(-1), the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L(-1), four other fractions (F2-F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L(-1)). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.
酵母抽提物在促进昆虫细胞生长和提高重组蛋白表达方面非常有效，因此它是无血清昆虫细胞培养基配方的关键组成部分。然而，酵母抽提物是一种复杂的混合物，其促进细胞生长的成分尚未得到鉴定。本研究采用连续乙醇沉淀法将酵母抽提物超滤物（YUF）分为六个馏分（F1-F6）。对各馏分进行了特征分析，并评价了其促进生长的活性。F1 是通过 65%乙醇沉淀得到的。当以 1 g/L 的浓度添加到 IPL-41 培养基中时，F1 与 YUF（在 IPL-41 培养基中 1 g/L）相比，可使 Sf-9 细胞的生长提高 71%，β-半乳糖苷酶产量提高 22%。然而，随着 F1 在 IPL-41 培养基中的浓度增加，其促进细胞生长的优势逐渐减弱。在 4 g/L 时，F1 的相对活性为 93%，而 YUF 在相同浓度下为 100%。在 1 g/L 时，另外四个用较高乙醇浓度沉淀的馏分（F2-F5）以及最终的上清液 F6 的生长促进活性与 YUF（100%）相比为 32-80%。有趣的是，当 F6 在存在高浓度 F1（>3 g/L）的情况下添加到 IPL-41 培养基中时，观察到促进细胞生长的协同作用。结果表明，乙醇沉淀是从 YUF 中分离促进生长成分的一种实用方法，但有多个成分共同促进 Sf-9 细胞的最佳生长。需要进一步的分离、分离和鉴定各个活性成分，以更好地了解这些成分对细胞代谢的作用。
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