New Proteins with Biomedical Activity
Engineering and investigation of de novo proteins carrying important biomedical activities
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Institute of Bioorganic Chemistry, Russia, Moscow
- University of Pennsylvania / Fox Chase Cancer Center, USA, PA, Philadelphia\nScripps Research Institute, USA, CA, La Jolla\nMedical Research Council / Laboratory of Molecular Biology, UK, Cambridge\nStanford University, USA, CA, Stanford\nHarvard University / Department of Chemistry and Chemical Biology, USA, MA, Cambridge\nCelera Genomics, USA, MD, Rockville\nUniversity of California / Department of Chemistry and Biochemistry, USA, CA, Santa Cruz\nCalifornia State University Northridge / Department of Chemistry and Biochemistry, USA, CA, Long Beach\nCLONTECHLaboratories, Inc. / BD Biosciences, USA, CA, Palo Alto
Project summaryThe main goal of the project is to obtain de novo proteins possessing biological activities of applied medical or biotechnological interest. Design of proteins with desirable structure and activity is one of the most intriguing challenges of modern protein engineering. Two main motivations for the de novo design are (i) complete understanding of principles of protein structure which is impossible without our ability to create artificial proteins similar to the natural ones; and (ii) de novo design is the essential first step toward constructing novel macromolecules which may solve important medical and biological problems. Initially the designers' interests were focused on structural (theoretical) aspects of de novo design, namely, on design of proteins with pre-designed structure. Our group have obtained and investigated first de novo protein [1,2] possessing not only desirable structure, but a model biological activity on murine thymocyte cells. The protein called albeferon was constructed basing on a de novo protein albebetin  with a pre-designed structure and an active octapeptide fragment from human interferon 2. It was shown that albeferon bound to cell receptors and efficiently activated blast-transformation of murine thymocytes [1,2]. Thus it was demonstrated that, in principle, de novo proteins might be used as carriers of biological activities. We believe that the next logical step in the field will be the constructing of de novo proteins carrying biological activities of applied medical and biotechnological interest. We have shown  that alblbetin (as well as albeferon) possessed low immunogenity therefore it might serve as a unique carrier which would not provoke strong immunic response. Such protein constructs based on albebetin will possess useful biological and pharmacological properties and may be used for engineering of new effective pharmaceuticals and biocompatible products. In this project we propose to engineer and investigate in detail several de novo proteins carrying active fragments corresponding to biological activities of high biomedical interest (anti-viral, anti-tumor, etc.) based on albebetin. The project includes the following research milestones.
(i) Design of de novo proteins carrying active fragments;
(ii) Engineering of the corresponding genes and expressing plasmids;
(iii) Production of the proteins in preparative amounts;
(iv) Study of their physical and chemical properties;
(v) Biological study of the obtained proteins;
(vi) Analysis of the proteins as possible pharmaceuticals including immunological tests and pre-clinical trials.
A set of modern research techniques are planned to be applied to resolve the problems set in the project, namely, computer molecular modeling, PCR-based mutagenesis and other methods of genetic engineering, gene expression in fusion expression systems, liquid chromatography techniques including affinity chromatography for protein purification, physical methods of protein investigation including circular dichroism and fluorescence, size-exclusion chromatography, urea-gradient electrophoresis, and other techniques giving general structural information on the proteins. Radioligand-binding assay will be applied to study receptor-binding properties of the obtained proteins. Functional activities of the proteins and their immunological properties will be studied using specific biological assays and antigenic mapping by NCP technique. For detailed study of the most interesting protein(s) we will use nuclear magnetic resonance. The project team has all mayor equipment and facilities necessary for the implementation of the planned research.
The project implies tight co-operation between two Russian research groups, which include qualified investigators possessing complementary experience in the field of the project. Scientists from IBC are highly experienced in protein engineering while researchers from the IIE have profound knowledge in biological investigation of active peptides. The project team are engaged in de novo protein design since 1991 [3,5]. The American collaborator, Dr. H. Roder and his laboratory have unique experience in protein folding and investigation of de novo proteins [6,7]. Contacts with this laboratory will will give to Russian scientists the possibility to improve their scientific level and to apply the accumulated experience to peaceful fundamental and applied research. As a whole the project will provide to weapon scientists and engineers from both Russian Institutes the opportunity for civil scientific and research activity thus responding to the primary objectives of the ISTC.
The project will give results of theoretical as well as practical value. The theoretical value is that engineering and investigation of new active de novo proteins will result in better understanding of basic principles of protein structure and structure-functional relationship in proteins. De novo proteins carrying important biological activities can be used also as convenient models to study these activities and their interaction with the carrier de novo proteins. Original data on the properties of these active protein constructs will permit to elaborate novel scientifically justified methods of engineering a potential protein medicines. Practical value of this project is that the obtained protein constructs will possess useful biological and pharmacological properties and may be used as new-type potential medicines. Thus, one could decrease side effects of natural pharmaceutical proteins which carry many activities using only the biological activity of interest grafted to a de novo protein. On the other hand, an active peptide fragment included into de novo protein will be more stable than the peptide in unbound form. De novo proteins designed from the basic structural principles as frameworks for isolated biological activities would possess lower immunogenity than original natural proteins carring these activities because high immunogenic T- and B-cell epitopes may be excluded.
We have analyzed a number of possible biomedical activities to graft to albebetin and selected several activities as preliminary candidates for grafting. These activities mediated by short peptides from amino acid sequences of human interferon б2, insulin and differentiation factor. We are looking also for other active fragments of medical or biotechnological importance described in the literature in order to introduce them into de novo protein. In the scope of this project we plan to design, obtain and investigate in detail at least 3-5 such biologically active protein constructs.
1. I.Yu.Aphasizheva, D.A.Dolgikh, Z.Kh.Abdullaev et al. (1998). FEBS Lett. 425:101-104.
2. D.A.Dolgikh, V.N.Uversky., A.E.Gabrielian et al. (1996). Protein Egng. 9:195-201.
3. A.N.Fedorov, D.A.Dolgikh, V.V.Chemeris et al. (1992). J. Mol. Biol. 225:927-931.
4. I.Yu.Aphasizheva, D.A.Dolgikh, M.P.Kirpichnikov (1999). Mol. Biol. (Mosk.) 33:679-683.
5. D.A.Dolgikh, A.N.Fedorov, V.V.Chemeris et al. (1991). Dokl. Akad. Nauk SSSR. 320:1266-1270
6. S.-H.Park, M.C.R.Shastry, H. Roder (1999). Nature Struct. Biol. 6:943-947.
7. S.T.Walsh, H.Cheng, J.W.Bryson et al.(1999). Proc. Natl. Acad. Sci. U S A. 96:95486-95491