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使用固定化RNA酶的亲和色谱法。

Affinity chromatography with an immobilized RNA enzyme.

作者信息

Vioque A, Altman S

出版信息

Proc Natl Acad Sci U S A. 1986 Aug;83(16):5904-8. doi: 10.1073/pnas.83.16.5904.

Abstract

M1 RNA, the catalytic subunit of Escherichia coli RNase P, has been covalently linked at its 3' terminus to agarose beads. Unlike M1 RNA, which is active in solution in the absence of the protein component (C5) of RNase P, the RNA linked to the beads is active only in the presence of C5 protein. Affinity chromatography of crude extracts of E. coli on a column prepared from the beads to which the RNA has been crosslinked results in the purification of C5 protein in a single step. The protein has been purified in this manner from cells that contain a plasmid, pINIIIR20, which includes the gene that codes for C5 protein. A 6-fold amplification of the expression of C5 protein is found in these cells after induction as compared to cells that do not harbor the plasmid.

摘要

M1 RNA是大肠杆菌核糖核酸酶P的催化亚基,已在其3'末端与琼脂糖珠共价连接。与在没有核糖核酸酶P的蛋白质组分(C5)时在溶液中有活性的M1 RNA不同,与珠子相连的RNA仅在C5蛋白存在时才有活性。用交联了RNA的珠子制备的柱子对大肠杆菌粗提物进行亲和层析,可一步纯化C5蛋白。已通过这种方式从含有质粒pINIIIR20的细胞中纯化出该蛋白,该质粒包含编码C5蛋白的基因。与不含该质粒的细胞相比,诱导后这些细胞中C5蛋白的表达扩增了6倍。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4886/386405/ed52039503c4/pnas00320-0161-a.jpg

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