Research project to improve the conformation and physical stability of proteins with high therapeutic values.
Mohammed Mahmoud Shawky Kassab, molecular biologist, specialist of microbiology, immunology, pharmacology and toxicology, faculty of pharmacy, Cairo University, Egypt.
1. Isolation, screening and identification of enzyme producing bacterial species using selective media.
2. Separation of the gene of the therapeutic protein of interest using suitable endonuclease enzymes, insertion of it in a suitable vector for its expression such as Saccharomyces cerevisiae using ligase enzyme.
3. Purification of the enzyme of interest from the supernatant of centrifuge tube as extracellular protein by crystallization by ammonium sulfate.
4. Characterization of the optimal physiological and environmental conditions of enzyme production and activity by biochemical and special chemical tests for the protein of interest.
5. Screening of therapeutic effect and determination of optimal dose for biological activity through animal models.
6. Determination, and formulation of a suitable dosage form and route of administration such as intramuscular or intravenous injections.
7. Identify the most flexible regions of the gene of therapeutic protein using conformational entropy and bioinformatics genetic engineering application software.
8. Induction of a mutation by single nucleotide polymorphism in the flexible region of alpha helices of the core of the protein to substitute two residues of flexible amino acids such as glycine with two cysteine residues adjacent to each other and are not apart by more than 0.1 nm from each other to form intermolecular and intramolecular disulfide bonds which increase the physical stability and the conformation of the protein of interest.
9. Insertion of the mutated gene of protein of interest in a suitable expression vector such as Eschrechia coli or Saccharomyces cerevisiae which secrets the protein of interest extracellulary.
10.After centrifugation for few minutes, purify and precipitate the modified and mutated extracellular therapeutic protein from the supernatant of centrifuge tube by crystallization by ammonium sulfate.
11.Screening of physical stability and identification of optimal biological activity of the modified and mutated therapeutic protein through animal models and tissue culturing testing.