Alexey N. Fedorov
The basic goal of our research is development of new systems for producing and stabilization of proteins and methods of their affinity purification. We use molecular chaperones as a framework for making fusion systems to improve characteristics of target protein and efficiently produce hard-to-express target polypeptides.
Recently we have developed a protein fusion system with minichaperone as a carrier to produce hydrophobic insoluble proteins in soluble stable forms. The system takes advantage of minichaperone, the apical domain of bacterial chaperone GroEL, which retains ability to bind polypeptide substrates and in some cases promote their folding.
We have demonstrated with two unrelated insoluble proteins that in the fusion complexes they become soluble and stable in solution at high concentrations for prolonged periods of time. For protein affinity purification, we focus on His-tag system.
We have developed a method for efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography. The project is in progress on making new His-tags with improved patterns.
- Fedorov, A., Dolgikh, D., Chemeris, V., Chernov, B., Finkelshtein, A., Schulga, A., Alakhov, Yu., Kirpichnikov, M. & Ptitsyn, O. De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level. — J. Mol. Biol., 1992, v. 225, p. 927-931
- Fedorov A., Baldwin T. Contribution of cotranslational folding to the rate of formation of native protein structure. — Proc. Natl. Acad. Sci. USA, 1995, v. 92, p. 1227-1231
- Fedorov A., Baldwin T. Cotranslational protein folding. — J. Biol. Chem., 1997, v. 272, p. 32715-32718
- Fedorov A., Baldwin T. Protein folding and assembly in a cell-free expression system. — Methods in Enzymology, 1998, v. 290, p. 1-17
- Fedorov A., Baldwin T. Process of biosynthetic protein folding determines the rapid formation of native structure. — J. Mol. Biol., 1999, v. 268, p. 712-723
- Sharapova O.A., Laurinavichyute D.K., Yurkova M.S., Andronova S.M., Fedorov A.N., Severin S.E., Severin E.S. Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography. — Journal of Chromatography, 2011, 1218(31): 5115-9