Dainiak Maria B, Plieva Fatima M, Galaev Igor Yu, Hatti-Kaul Rajni, Mattiasson Bo
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Biotechnol Prog. 2005 Mar-Apr;21(2):644-9. doi: 10.1021/bp049615g.
Supermacroporous monolithic columns with Cu(2+)-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10-100 microm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the Cu(2+)-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
带有Cu(2+)-IDA配体的超大孔整体柱已成功用于不同类型微生物细胞的色谱分离。整体基质床由与亚甲基双丙烯酰胺交联的聚丙烯酰胺冷冻凝胶形成，具有大（10 - 100微米）的相互连接的孔网络，允许全细胞在不含配体的普通冷冻凝胶柱中不受阻碍地通过。研究了两个模型系统：野生型大肠杆菌（w.t. E. coli）与展示多组氨酸肽的重组大肠杆菌细胞（His标记的大肠杆菌）的混合物，以及w.t. E. coli与嗜碱芽孢杆菌细胞的混合物。从含有等量两种细胞类型的原料中定量捕获野生型大肠杆菌和His标记的大肠杆菌，并通过用咪唑和EDTA选择性洗脱进行回收，产率分别为80%和77%。与主要含有弱结合的野生型大肠杆菌细胞的咪唑洗脱峰相比，EDTA洗脱后获得的峰中His标记的大肠杆菌细胞富集了8倍。嗜碱嗜盐的嗜碱芽孢杆菌细胞对Cu(2+)-IDA冷冻凝胶柱的亲和力较低，可以与野生型大肠杆菌细胞的混合物有效分离，野生型大肠杆菌细胞通过咪唑梯度从柱中高效保留并回收。所有细胞在色谱过程后均保持其活力。结果表明，亲和超大孔整体柱色谱是一种有效分离单个细胞类型的有前途的方法。
