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Feb 1, 2009 (Vol. 29, No. 3)

Promega's Protein Purification Project

  • Promega recently introduced a couple of new tools to make life easier for recombinant protein aficionados. HaloTag, despite a name that conjures up cherubim and seraphim, is a solid, down-to-earth product. According to Kate Qin Zhao, Ph.D., a senior research scientist in proteomics R&D, the HaloTag concept was originally based on a bacterial dehalogenase, which will covalently attach to a set of chloroalkane ligands with different functional groups, such as fluorescent dyes, biotin, and solid surfaces. By forming a fusion protein with a gene product of interest, it can perform as a powerful tag and purification device. According to Dr. Zhao, it has been successfully applied to a variety of biotech tasks, such as live-cell imaging, protein interactions, and protein immobilization.

    Now a next-generation HaloTag 7 has been configured in order to increase its structural compatibility with its numerous fusion protein partners. The newly redesigned model boasts a number of desirable features, including superior performance compared to GST, MBP and His-tags with better protein recovery, and lower nonspecific contaminants.

    Fusion tags are frequently introduced to facilitate protein purification of recombinant proteins. Removal of the tag after purification is usually done by engineering a cleavage site between the tag and the encoded protein recognizable by a site-specific protease, such as the one from tobacco etch virus (TEV). Promega scientists have introduced a TEV linker that allows the protein of interest to be efficiently cleaved from the HaloTag 7 protein, greatly improving the quality of purification. So the system produces proteins with superior yield, purity, and specific activity by providing a simple method for fusion protein detection and quantification. The HaloTag protein purification system will be released this spring.

    Dr. Zhao also discussed two cell-free protein expression systems, the S30 T7 High Yield Protein Expression System and the TNT® T7 Insect Cell Extract Protein Expression System. She claims they offer a number of important advantages. The prokaryotic S30 System is a native coupled-transcription/translation device that features high protein productivity per DNA input, with yields as high as 500 µg/mL/hr at 37ºC. Because of its ease of manipulation, it can be used as an efficient screening tool. It is scalable and as little as 5 µL reaction mixtures can be driven with 100 ng plasmid DNA. And the system has the flexibility to permit use of multiple detection methods, including Coomassie, FluoroTect™, Transcend™, HaloTag®Protein purification and His/HQ tagHaloTag.

    Finally, the TNT T7 Insect Cell Extract Protein Expression System, designed as a eukaryotic system, uses a DNA template with a T7 promoter for protein yields up to 75 µg/mL. “We believe it offers a higher probability of obtaining proteins with high specific activity,” states Dr. Zhao.



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