Author:
Yurkova Maria S.,Sharapova Olga A.,Zenin Vladimir A.,Fedorov Alexey N.
Abstract
Abstract
Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.
Publisher
Springer Science and Business Media LLC
Reference55 articles.
1. Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523–533 (2003).
2. Riggs, P., La Vallie, E. R. & McCoy, J. M. Introduction to expression by fusion protein vectors. Curr. Protoc. Mol. Biol. Ed. Frederick, M. Ausubel, A. l. Chapter 16, Unit16.4A (2001).
3. Waugh, D. S. Making the most of affinity tags. Trends Biotechnol. 23, 316–320 (2005).
4. Schmidt, S. R. Fusion-proteins as biopharmaceuticals–applications and challenges. Curr. Opin. Drug Discov. Devel. 12, 284–295 (2009).
5. Esposito, D. & Chatterjee, D. K. Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. Biotechnol. 17, 353–358 (2006).
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