Technical Highlight - February 2014
Short description: A new series of protein expression vectors with 'built in' tRNA genes that recognize rare codons promises to accelerate and enhance the determination of novel protein structures.
Whole-genome sequencing initiatives have rapidly expanded the number of promising candidate proteins for structural analysis and functional studies. Yet, variations in codon usage among source organisms pose an inherent technical challenge to potentially enlightening comparative studies. A tRNA that recognizes a codon triplet preferred by the source organism may be rare in Escherichia coli, preventing production of full-length target proteins at levels sufficient for structural determination.
This obstacle is most commonly circumvented by cotransforming a second plasmid construct bearing the low-abundance tRNA genes into the expression strain. Now, Joachimiak and colleagues (PSI MCSG) have streamlined the LIC vectors that were designed for high-throughput (HTP) expression by the Midwest Center for Structural Genomics, and have introduced E. coli tRNA genes for rare Ile (AUA) and Arg (AGG/AGA) codons. The result is a series of ten expression vectors that are entirely compatible with the MCSG's established HTP cloning and production protocols, with additional features to facilitate structural investigations.
In the new series of vectors, the cleavable His6 affinity purification tag of the original pMCSG7 vector was retained, and 1kb of nonessential plasmid sequence was removed to accommodate long coding sequences or multiple gene insertions. The authors demonstrate high-yield coexpression of six proteins from a single construct containing dual insertion sites, making this system uniquely suited for coexpression and purification of multisubunit complexes. Other members of this vector series additionally include a biotinylation sequence and a gene encoding the BirA ligase to direct production of biotinylated, His-tagged target proteins. Biotinylated fusion proteins produced in vivo can be directly immobilized for ligand screening or binding affinity studies, and the authors show that semiautomated methods such as biolayer interferometry can be used to determine binding constants of fusion proteins that have been purified by established HTP protocols.
The enhanced protein yields provided by the tRNA gene integration and the expanded cloning, purification and selection features of these new pMCSG expression vectors should find widespread use among the structural genomics community, and are available through the PSI:Biology Materials Repository.
W.H. Eschenfeldt et al. New LIC vectors for production of proteins from genes containing rare codons.
J Struct Funct Genomics. 14, 135-144 (2013). doi:10.1007/s10969-013-9163-9