New LIC vectors for production of proteins from genes containing rare codons.
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 coexpressed 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.
Biosciences Division, Midwest Center for Structural Genomics, Argonne National Laboratory, Bldg. 202/Rm. BE111, 9700 South Cass Avenue, Argonne, IL, 60439, USA.
William H
WH
Eschenfeldt
Magdalena
M
Makowska-Grzyska
Lucy
L
Stols
Mark I
MI
Donnelly
Robert
R
Jedrzejczak
Andrzej
A
Joachimiak
D019921:Biotinylation; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D007700:Kinetics; D008024:Ligands; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D003062:Codon; D008024:Ligands; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012343:RNA, Transfer