Proteome analysis of animal cells for development of efficient serum-free culture technology

动物细胞的无血清培养对于临床和诊断上重要的蛋白质的大规模生产是必不可少的。为了开发高效的无血清培养技术，我们对中国仓鼠卵巢（CHO）细胞进行了蛋白质组学分析，CHO细胞被广泛用作生产复杂重组蛋白的宿主细胞。通过将维持在补充有10%胎牛血清（FBS）的培养基中的细胞逐步适应于含有较低浓度血清的培养基并最终适应于无血清培养基，建立能够在无血清培养基中生长的CHO细胞。测定了适应于无血清培养生长的细胞的比生长速率和比重组蛋白生产速率，发现其与维持在FBS补充培养基中的CHO细胞的比生长速率和比重组蛋白生产速率相当。然后通过细胞内蛋白质的二维凝胶电泳来研究血清非依赖性细胞，其中在第一维中具有固定的pH梯度（pH 3-10），在第二维中具有线性丙烯酰胺梯度（8-18%T）。将所得蛋白质表达模式与血清依赖性细胞中的蛋白质表达模式进行比较。在分析的700多个蛋白质点中，有几个蛋白质点显示出明显的增加或减少，表明这些蛋白质可能在无血清培养中的生长中起重要作用。

Serum-free culture of animal cells is essential for large-scale production of clinically and diagnostically important proteins. With the aim of developing efficient serum-free culture technology, we have undertaken a proteomic analysis of Chinese hamster ovary (CHO) cells, which are widely used as host cells in the production of complex recombinant proteins. CHO cells capable of growing in a serum-free medium were established by stepwise adaptation of cells maintained in a medium supplemented with 10% fetal bovine serum (FBS) to a medium containing lower concentrations of serum and finally to a serum-free medium. Specific growth rate and specific recombinant protein production rate of the cells adapted to growth in serum-free culture were measured and found to he comparable to those of CHO cells maintained in a FBS-supplemented medium. The serum-independent cells were then studied by two-dimensional gel electrophoresis of intracellular proteins with immobilized pH gradient (pH 3-10) in the first dimension and a linear acrylamide gradient (8-18%T) in the second dimension. The resulting protein expression patterns were compared with those in serum-dependent cells. Among over 700 protein spots analyzed, several spots showed marked increase or decrease, indicating that these proteins may play an important role in growth in serum-free culture.