Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes
Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes
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DOI：
10.1006/prep.2000.1313
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发表时间：
2000-12-01
影响因子：
1.6
通讯作者：
Marmorstein, R
中科院分区：
文献类型：
--
作者：
Johnston, K;Clements, A;Marmorstein, R
本报告描述了一种双载体共表达系统的开发和应用，用于在细菌中过量生产异聚体细胞周期和转录调控蛋白复合物。为了便于这些研究，我们构建了一个基于T7的表达质粒，pRM 1，它含有一个来自p15 A的复制起点和一个编码卡那霉素抗性的基因。该表达载体与T7表达载体的pET家族中发现的ColE 1衍生的质粒相容，所述质粒编码氨苄青霉素抗性。它还具有与pET衍生的pRSET载体相同的多克隆位点，允许在两种表达载体之间容易地穿梭。将pRM 1和pET衍生的表达载体共转化到大肠杆菌菌株如BL 21（DE 3）中导致异聚蛋白复合物的显著水平的共表达。我们证明了将pRM 1和pET衍生载体结合用于细胞周期调控组分pRB/E7和pRB/E1 a以及转录调控复合物SRF/SAP-1和SRF/Elk-1的共表达的适用性。我们进一步使用pRB/E1 a复合物来证明这些共表达的复合物可以被纯化至均一以用于进一步的研究。与pET衍生的载体组合使用的pRM 1载体通常应适用于大规模共表达和纯化各种异聚蛋白复合物，用于生物化学、生物物理和结构研究。(C)北京大学出版社.
This report describes the development and application of a dual vector coexpression system for the overproduction of heteromeric cell cycle and transcriptional regulatory protein complexes in bacteria. To facilitate these studies we constructed a T7-based expression plasmid, pRM1 that contains an origin of replication derived from p15A, and a gene encoding kanamycin resistance. This expression vector is compatible with ColE1-derived plasmids found in the pET family of T7 expression vectors, which encode ampicillin resistance. It also has the same multiple cloning sites as the pET- derived pRSET vector, allowing easy shuttling between the two expression vectors. Cotransformation of the pRM1 and pET-derived expression vectors into an Escherichia coli strain such as BL21(DE3) results in a significant level of coexpression of heteromeric protein complexes. We demonstrate the applicability of combining the pRM1 and pET-derived vectors for the coexpression of cell cycle regulatory components, pRB/E7 and pRB/E1a, and the transcriptional regulatory complexes, SRF/SAP-1 and SRF/Elk-1. We further use the pRB/E1a complex to demonstrate that these coexpressed complexes can be purified to homogeneity for further studies. Use of the pRM1 vector in combination with the pET-derived vectors should be generally applicable for the large-scale coexpression and purification of a wide variety of heteromeric protein complexes for biochemical, biophysical, and structural studies. (C) 2000 Academic Press.