Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy.
Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2-Msh6 mismatch repair protein (248kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function.
Finkelstein
Jeff
J
Rockefeller University and Howard Hughes Medical Institute, New York, NY 10021, USA.
Antony
Edwin
E
Hingorani
Manju M
MM
O'Donnell
Michael
M
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D046911:Macromolecular Substances; D008954:Models, Biological; D051718:MutS Homolog 2 Protein; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C098790:MSH6 protein, S cerevisiae; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein