Maximum IMPACT: The IMPACT T7 System for Recombinant Protein Expression and Purification

Protein yield and ease of purification are two important criteria in choosing a system for the expression/ purification of recombinant proteins. The IMPACT (Intein Mediated Purification with an Affinity Chitin- binding Tag) T7 System from New England BioLabs, Inc., enables the purification of recombinant fusion proteins in a single chromatographic step. A major advantage of the IMPACT system is that a self-cleavage reaction is used to release the affinity tag from the protein of interest. Thus, proteases are not required to separate the target protein from the affinity tag. In other commercially available expression/purification systems, the use of proteases necessitates additional purification steps and can also be associated with other difficulties (such as cleavage at secondary sites). Schematic illustration of the IMPACT T7 purification system. Source: Copyright 1996/97 New England BioLabs Catalog. Reprinted with permission. New England BioLabs' IMPACT system utilizes a modified protein splicing element (intein) in conjunction with a chitin-binding domain (CBD) as an affinity tag (S. Chong et al., Gene 192: 271-281, 1997). The CBD portion is used to purify the fusion protein via affinity chromatography, and the intein enables subsequent separation of the target protein by the self-cleavage reaction. Protein purification is achieved by one chromatographic procedure. The target protein-intein-CBD fusion is first bound to a chitin affinity column. While still bound to the chitin column, the target protein is cleaved from the intein-CBD affinity tag. The column is simply equilibrated at 4°C with a buffer containing a thiol (such as dithiothreitol, ß-mercaptoethanol, or cysteine). Under these conditions, intein undergoes self-cleavage at its N-terminal peptide linkage. After the self-cleavage reaction, the protein of interest is eluted, and the intein-CBD fusion remains bound to the column. Protein expression is driven from a tac promoter in IMPACT I, New England BioLabs' first product to exploit the intein technology. This product achieved some popularity in the United States, as well as abroad. "I think that the IMPACT system is the most elegant protein-purification system so far," says Uli Schmidt, a graduate student at the Institut für Biotechnologie, Halle, Germany. Mr. Schmidt used IMPACT I to purify a pentameric viral coat protein but added that protein yields were quite low using this method. Recently, the tac-based IMPACT I system was replaced with IMPACT T7, which uses a T7 promoter. For many proteins, higher levels of protein expression are obtained using the T7-based system. Dr. Joanna Porankiewicz, a guest researcher at the Plant Physiology Department of Umeå University, Sweden, has used IMPACT I extensively and is now conducting studies using IMPACT T7. She says, "I am personally very enthusiastic about the IMPACT system. I found it easy and convenient....The eluted protein was in our case almost 90-95 percent pure." Dr. Ming-Qun Xu, a senior scientist at New England BioLabs, is optimistic about the current applications of IMPACT but notes that additional work addressing different application-specific technical problems is ongoing. For example, dithiothreitol is known to have adverse effects on some eukaryotic proteins. To circumvent this problem, researchers at New England BioLabs are currently optimizing the use of pH shifts as an alternative method for the induction of the intein-mediated cleavage reaction. Dr. Xu adds, "The IMPACT system represents the first generation...of intein-based products. I don't expect the current vectors (or any system) to work for all proteins. Like the complexity of the protein world, we also need diversified product lines for protein purification." For more information, visit New England BioLabs' website at www.neb.com, or call 800-632-7799. The author is Deborah Wilkinson, a freelance writer based in Memphis, Tenn. She may be contacted at deborahwilkinson@sprynet.com.

Maximum IMPACT: The IMPACT T7 System for Recombinant Protein Expression and Purification

The Scientist Staff

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