Technical Highlight - November 2012
Short description: A tag variation strategy enables increased membrane protein production.
Membrane proteins such as G protein-coupled receptors (GPCRs) are infamously difficult to express, and much effort has been dedicated to developing cell-free expression systems and optimizing recombinant protein production. One tripping point involves translation initiation, where the formation of secondary structures in the mRNA 5′ end can impede ribosome binding. This has been overcome for some proteins by tagging targets with 5′ sequences, including those from outer membrane protein A and green fluorescent protein (GFP) genes.
Based on these successes, Bernhard and colleagues (PSI MPSbyNMR)established a tag variation strategy to optimize production of low-expressing targets in a cell-free system. To minimize the effect on protein structure, they designed small, 6–8 residue tags, followed by a PreScission protease cleavage site. The seven tags driven by the T7 promoter with a subsequent ribosome-binding site ranged from a tag with 100% AT content, which forms less stable secondary structures, to one based on the commonly used hemagglutinin tag and another based on the first codons of GFP, as well as a random sequence tag and a longer, previously used T7 tag.
The authors optimized the construction strategy by using common primers. Comparison of expression efficiencies in the cell-free system using linear and vector templates revealed that the former can be used directly without compromising yield—an additional time and cost savings.
As proof of concept, the group applied this strategy to a set of GPCRs. While tagging only moderately increased expression of reasonably well-expressed proteins, substantial improvement was achieved for GPCRs that were otherwise poorly expressed. Most impressive was the improvement from 0.08 to 2.33 mg/ml for glucagone-like peptide 1 receptor. For different proteins, best expression was achieved with different tags, and based on the results, four expression tags (the AT-tag, the H-tag, the G-tag and the SER-tag) are recommended for screening.
Tagged GPCRs produced using this system had expected α-helical folds and were of sufficient quality. Going further, the authors optimized the best tag by removing the PreScission site and subsequently truncating the tag. This approach should prove beneficial for the production of this important class of proteins.
S. Haberstock et al. A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems.
Protein Expr. Purif. 82, 308-316 (2012). doi:10.1016/j.pep.2012.01.018