title: Coexpression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes.
abstract: 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.
authors.affiliation: The Wistar Institute, Philadelphia, Pennsylvania, 19104, USA.
authors.forename: K
authors.initials: K
authors.lastname: Johnston
authors.forename: A
authors.initials: A
authors.lastname: Clements
authors.forename: R N
authors.initials: RN
authors.lastname: Venkataramani
authors.forename: R C
authors.initials: RC
authors.lastname: Trievel
authors.forename: R
authors.initials: R
authors.lastname: Marmorstein
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D009687:Nuclear Proteins / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D026362:Serum Response Factor
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor