Title

Overproduction and Analysis of Eukaryotic Multiprotein Complexes in Escherichia coli Using a Dual-vector Strategy

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

8-2003

Publisher

Elsevier

Source Publication

Analytical Biochemistry

Source ISSN

0003-2697

Abstract

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 (248 kDa) 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.

Comments

Analytical Biochemistry, Vol. 319, No. 1 (August 2003): 78-87. DOI.

Edwin Antony was affiliated with Wesleyan University at time of publication.