Brissette L, Cahuzac-Bec N, Desforges M, Bec J L, Marcel Y L, Rassart E
Laboratoire du Métabolisme des Lipoprotéines, Institut de Recherches Cliniques de Montréal, Québec, Canada.
Protein Expr Purif. 1991 Aug;2(4):296-303. doi: 10.1016/1046-5928(91)90086-x.
Human recombinant apolipoprotein (apo) A-I was produced by Chinese hamster ovary (CHO) cells and Escherichia coli with expression vectors containing cDNAs encoding preproapoA-I or apoA-I, respectively. The apoA-I from CHO cells was purified from the culture medium by ammonium sulfate precipitation, phenyl-Sepharose chromatography, and affinity purification on anti-apoA-I immunoabsorber. Human apoA-I was produced in E. coli as a fusion protein with glutathione S-transferase. A four amino acid linker, which separated the two proteins, was specifically recognized and cut by Factor Xa. The purification was accomplished by chromatography of E. coli extracts on glutathione-Sepharose and digestion with Factor Xa. The highest production level was found to be 0.5 micrograms/ml of culture medium per 48 h for a clone of stable transformant of CHO cells, whereas E. coli could produce as much as 20 micrograms/ml of bacterial culture. These apoA-I forms were compared in terms of molecular weight, isoelectric point, and expression of several epitopes. Recombinant apoA-I obtained from CHO cells appears intact and its isoelectric point is compatible with that of the mature form and the proform of apoA-I, whereas a part of the apoA-I produced by E. coli does not contain the COOH-terminus. Also, two of six epitopes are expressed to a greater extent in apoA-I obtained from E. coli than in apoA-I obtained from human plasma.
人重组载脂蛋白（apo）A-I分别由中国仓鼠卵巢（CHO）细胞和大肠杆菌产生，所使用的表达载体分别含有编码前原载脂蛋白A-I或载脂蛋白A-I的cDNA。来自CHO细胞的载脂蛋白A-I通过硫酸铵沉淀、苯基琼脂糖层析以及在抗载脂蛋白A-I免疫吸附剂上进行亲和纯化，从培养基中分离出来。人载脂蛋白A-I在大肠杆菌中作为与谷胱甘肽S-转移酶的融合蛋白产生。一个四氨基酸接头将这两种蛋白分隔开，该接头可被因子Xa特异性识别并切割。纯化过程通过大肠杆菌提取物在谷胱甘肽琼脂糖上的层析以及用因子Xa消化来完成。对于CHO细胞稳定转化子的一个克隆，发现最高产量水平为每48小时0.5微克/毫升培养基，而大肠杆菌每毫升细菌培养物可产生多达20微克。对这些载脂蛋白A-I形式在分子量、等电点以及几个表位的表达方面进行了比较。从CHO细胞获得的重组载脂蛋白A-I看起来是完整的，其等电点与载脂蛋白A-I的成熟形式和前体形式相符，而大肠杆菌产生的部分载脂蛋白A-I不含COOH末端。此外，六个表位中的两个在从大肠杆菌获得的载脂蛋白A-I中比在从人血浆获得的载脂蛋白A-I中表达程度更高。
