Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes

This report describes the development and application of a dual vector coex pression system for the overproduction of heteromeric cell cycle and transc riptional regulatory protein complexes in bacteria. To facilitate these stu dies we constructed a T7-based expression plasmid, pRM1 that contains an or igin of replication derived from p15A, and a gene encoding kanamycin resist ance. This expression vector is compatible with ColE1-derived plasmids foun d in the pET family of T7 expression vectors, which encode ampicillin resis tance. It also has the same multiple cloning sites as the pET- derived pRSE T vector, allowing easy shuttling between the two expression vectors. Cotra nsformation of the pRM1 and pET-derived expression vectors into an Escheric hia coli strain such as BL21(DE3) results in a significant level of coexpre ssion of heteromeric protein complexes. We demonstrate the applicability of combining the pRM1 and pET-derived vectors for the coexpression of cell cy cle regulatory components, pRB/E7 and pRB/E1a, and the transcriptional regu latory complexes, SRF/SAP-1 and SRF/Elk-1. We further use the pRB/E1a compl ex to demonstrate that these coexpressed complexes can be purified to homog eneity for further studies. Use of the pRM1 vector in combination with the pET-derived vectors should be generally applicable for the large-scale coex pression and purification of a wide variety of heteromeric protein complexe s for biochemical, biophysical, and structural studies. (C) 2000 Academic P ress.