Protein Production Technology
Team: Ji Qiu, Ph.D., Justin Saul, and Joshua LaBaer, MD, Ph.D.
Purified proteins have innumerable uses in research, and, consequently, there is a great demand for high quality pure and active proteins. The most common protein expression host, E. coli, typically doesn’t work well for mammalian proteins. Full length mammalian proteins expressed in E. coli are usually insoluble. Sophisticated bioinformatics analysis is required to predict protein domains that express well in E. coli. Even when expressed, proper post-translational modifications often required for the proper folding or activity of eukaryotic proteins will not be present in a bacterial based system. On the other hand, expression and purification of human proteins in eukaryotic cells is cumbersome and costly. Therefore, it is quite challenging to produce high quality eukaryotic proteins for biomedical research.
We have designed a mammalian protein production pipeline for full-length proteins that utilizes recently developed in vitro protein expression systems. We typically employ multiple expression systems to achieve a high (>80%) success rate using E. coli, cell-free wheat germ extract (WGE) or cell-free human HeLa cell extract (HCE)(see diagram at right). We have adapted a HALO-tag based protein purification protocol followed by protease cleavage to ensure high purity. This pipeline has been automated with liquid handling robots, allowing for the purification of 96 proteins in parallel. Yields of purified proteins range from several to 10s of micrograms. We have successfully applied proteins produced from our system to the discovery and characterization of novel affinity reagents for the human proteome.