Spider Web Proteins for Medicine
Obtaining and Structural-Functional Investigations of Spider Web Recombinant Proteins and Their Synthetic Analogs for the Purpose of the Development of New Materials Designed for Medical Application
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Smirnova N V
State Scientific Center of Genetics and Selection of Industrial Microorganisms (GosNIIGenetica), Russia, Moscow
- Scientific Research Center "Uglechimvolokno", Russia, Moscow reg., Mytischi\nState Research Center for Applied Microbiology, Russia, Moscow reg., Obolensk
- University of Michigan/College of Pharmacy, USA, MI, Ann Arbor
Project summaryThe main goal of the Project is creation of yeast strains, producers of recombinant silk proteins analogs, investigation of their physical and chemical and bio-mechanical properties and investigation of potential of creation of medical materials on the basis of recombinant polypeptides.
At the first phase (June 1999 - May 2000) chemical-enzymatic synthesis was fulfilled of 400 bp gene (monomer) coded the fragment of dragline of Nephila clavipes spider spidroin 1. Using step by step duplication in multicopy vector dimer, tetramer, octamer and more high-molecular forms of monomer have been obtained. The genes obtained in vector composition have been transferred in yeast cells Saccharomices cerevisiae and their controlled expression was observed. The original methods were developed for recombinant protein testing as well as the methods for preparative extraction and refinement of these proteins from insoluble yeast cell fraction. Hundreds mg of tetrameric and octameric molecules were produced. These samples have been used for extensive physics-chemical studies. Tools for film formation on the basis of recombinant proteins have been elaborated with the aim of investigation of conformational transitions in fibrous proteins.
On the bases of results obtained an approach was suggested, which take into account interrelation between periodicity in amino acid location in fibrous protein sequences and protein properties.
The results obtained have good potential of providing a basis for new pharmaceuticals as well as for biologically compatible medical materials based on fibrous proteins (cover films for wound treatment, materials for substitution surgery, ligaments, etc)
– Obtained yeast strains producing recombinant silk protein analogs are expected to be of the important practical and potential commercial value.
– Obtained information about physical and chemical properties both of original spider silk proteins as well as modeling recombinant polypeptides will have theoretical significance for solving the fundamental problem of fibrous proteins structure-function relations.
– The development of the adequate approach for the modeling of fibrous proteins with necessary physical and chemical properties will have theoretical and practical importance.
– Laboratory testing on the animals of the samples of medical preparations created on the basis of new materials in film form, for the wound treatment.
– The possibility of the usage of the threads constructed from recombinant proteins as new materials for the surgery usage will be evaluated.
– The study of potential for design of new drugs and biocompatible materials on the basis of synthetic fibrous proteins will be performed.
The technical approach and methodology.
The oligonucleotides of the required sequence would be chemically synthesized on an automatic DNA synthesizer, which employs the phosphoramidite method. The monomers will be assembled from oligonucleotides by self-annealing and cloned in E.coli.
The following multimerization of the gene of interest would be performed in two ways:
– self-annealing and ligation of the purified monomers and introduction of the reaction products into bacterial cells followed by analyzing of recombinant DNAs from transformed bacterial cells;
– step by step duplication of the monomer in an initially obtained recombinant plasmid.
Various biochemical and protein chemistry techniques would be employed to develop new processes of extraction and purification of the recombinant spidroin-like protein. In particular, HPLC and other chromatography procedures could be mentioned.
Recombinant proteins would be studied with physical and chemical methods, which were previously successfully used to study natural dragline as well as natural and synthetic polymers. It is planed to use FTIR- spectroscopy, solid-state NMR spectroscopy, CD-spectroscopy, and NMR- relaxation.
Theoretical analysis of structural-functional relationships in recombinant proteins and their native analogs will be used.
The possibility of formation of polymeric films from recombinant polypeptides will be investigated in experiments with the solutions with various compositions.
The experiments on spinning solution formation and investigation of its properties will include solvent selection, investigation of rehological properties of the polypeptide solutions and research of the stream forming process and of precipitation conditions.
We are going to study the potential of new materials based on recombinant proteins and their analogs for medical applications, with the focus on therapeutic experiments on biological compatibility of the protein fibers with damaged and intact tissues of lab animals.