We have modified a series of LIC pMCSG vectors used in high-throughput protein production to include crucial tRNA genes covering rare codons for Arginine and Isoleucine.
In the effort to produce proteins coded by diverse genomes, structural genomics projects often must express genes containing codons that are rare in the production strain. To address this problem, genes expressing tRNAs corresponding to those codons are typically co-expressed from a second plasmid in the host strain, or from genes incorporated into production plasmids. Here we describe the modification of a series of LIC pMCSG vectors currently used in the high-throughput (HTP) production of proteins to include crucial tRNA genes covering rare codons for Arg (AGG/AGA) and Ile (AUA). We also present variants of these new vectors that allow analysis of ligand binding or co-expression of multiple proteins introduced through two independent LIC steps. Additionally, to accommodate the cloning of multiple large proteins, the size of the plasmids was reduced by approximately one kilobase through the removal of non-essential DNA from the base vector. Production of proteins from core vectors of this series validated the desired enhanced capabilities: higher yields of proteins expressed from genes with rare codons occurred in most cases, biotinylated derivatives enabled detailed automated ligand binding analysis, and multiple proteins introduced by dual LIC cloning were expressed successfully and in near balanced stoichiometry, allowing tandem purification of interacting proteins.
Vectors described in this paper can be ordered from the PSI:Biology-Materials Repository at http://psimr.asu.edu under the following accession codes: pMCSG53, pMCSG69, pMCSG58, pMCSG59, pMCSG70, pMCSG71, pMCSG62, pMCSG63, pMCSG76, and pMCSG77.
This research was the topic of an SBKB Technical Highlight, which can be found at http://sbkb.org/update/research/coaxing-rare-codons
The DyNA Vector map of the pMCSG69 empty vector showing some of the plasmid features, including the argU and ileX genes. For more information about this vector, please go to http://dnasu.org/DNASU/GetCloneDetail.do?cloneid=450865
Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, Jedrzejczak R, Joachimiak A. "New LIC vectors for production of proteins from genes containing rare codons." J. Struct. Funct. Genomics 14:135-44 (2013). doi: 10.1007/s10969-013-9163-9 PubMed ID: 24057978 | Search SBKB Publications portal
Andrzej Joachimiak, Email: firstname.lastname@example.org
Midwest Center for Structural Genomics
These LIC vectors can be ordered from the PSI:Biology-Materials Repository.
Last edited:Wed 26 Feb 2014 - 5 years, 3 months ago