ENGINEERED PROTEIN BY DNA TECHNOLOGY <br />PRESENTED BY:<br />M SANDEEP REDDY.<br />TRINITY COLLEGE OF PHARMACEUTICAL SCIENCES <br />H.NO: 11341P1033<br />M.Pharmacy (Pharmaceutics) 2 ndsemester<br />
DEFINITION<br />Protein engineering can be defined as the modification of protein structure with recombinant DNA technology or chemical treatment to get a desirable function for better use in medicine, industry and agriculture. <br />
Protein engineering is merging of several disciplines like: 1.molecular biology.2. protein chemistry.3. enzymology .4.structural chemistry to alter catalytic or structural stability of protein.<br />
BASIC ASSUMPTIONS <br />While doing protein engineering should recognize the following properties of ,<br />many amino acid substitution, deletions or additions lead to no changes in enzyme activity so that they are silent mutator.<br />Protein have limited number of basic structures and only minor changes are superimposed on them leading to variation<br />Similar patterns of chain folding and domain structure can arise from different amino acid sequences with little or no homology<br />
OBJECTIVES OF PROTIEN ENGINEERING<br />The objectives of protein engineering is as follows – <br />(a) to create a superior enzyme to catalyze the production of high value specific chemicals.<br />(b) to produce enzyme in large quantities.<br />(c) to produce biological compounds(include synthetic peptide, storage protein, and synthetic drugs) superior to natural one CONTINUE…<br />
Get humanised(chimeric) antibodies with less immunogenicity .<br />Make harmones resistant to attack by antibodies or stomach enzymes.<br />Get more site specific, more potent biopharmaceutical with altered pharmacological action.<br />Over all aim of protien engineering application is to get funtionally more useful enzymes, antibodies, harmones, receptor protiens.<br />
Steps involved in protein engineering<br />A study of three dimensional structure of protein <br /> A study of three dimensional structure is the preliminary steps of protein engineering. And a 3d structure of protein is produced from the data generated from X-ray crystallography and NMR process by protein modeling<br />
The three dimensional structure of a protein<br />
<ul><li>The continuous line represents the primary structure of the protein.
You should note that the primary structure has a polarity. That is, there is a N terminal region of the protein and a C terminal region.
The curly sections represent alpha helical regions. These are components of the secondary structure of a protein.
The flat sections with arrows represent beta pleated sheat. These are also components of the secondary structure of a protein. </li></li></ul><li>Methods for protein engineering<br />methods for protein engineering <br />Chemical modification<br />Genetic modification<br />
GENTIC MODIFICATION<br />In the gene modification method is to design, develop and produce proteins with improved operating characteristics ( increased stability & biological activity) sometimes creating even novel proteins. <br /> The techniques such as Mutagenesis( site-directed ).& gene cloning are utilized for this purpose.<br />
increased stability & biological activity<br /> Performed for thermo stability of proteins, their industrial application and therapeutic use can more appropriately met.<br /> It includes:<br /><ul><li>ADDITION OFSULIFIDE BONDS
SINGLE AMINO ACID CHANGES.</li></li></ul><li>ADDITION OF SULIFIDE BONDS<br />Significantly increase in the thermostability of enzymes. However , the addition al disulfide bonds should not interfere with the normal enzyme function.<br />The new protein with added disulfide bonds does not readily unfold at high temperatures.<br />Examples: T4-Lysozyme, Xylanase.<br />
CHANGE OF ASPARAGINE TO OTHER AMINO ACIDS<br />If asparagine and glutamine present in protein when heated, ammonia is released amino acids convert to aspartic acid and glutamic acid. Protein may refold and loss of biological activity. <br />Example: triosephosphateisomerase. <br />
REDUCING THE FREE SULFHYDRYL GROUPS<br />The protein or enzyme stability and its activity can be increased by reducing the number of sulfhydryl groups. <br />Convert Cys to another amino acid (serine?)<br /><ul><li> reduce dimerization.
maintain activity of enzyme.</li></ul>Examples: Human β- interferons<br />
SINGLE AMINO ACID CHANGES.<br />Some of the recombinant proteins can be improved in their stability and biology activity by a secondgeneration variants. These have been frequently achieved by a single amino acid changes. <br /> Examples: α1 Antitrypsin, insulin, tissue plasminogen activator, hirudin.<br />
The other process of gene modification are-<br />In vitro mutagenesis using synthetic oligonucleotides.<br />Synthesis of complete modified gene de novo<br />
(a) In vitro mutagenesis using synthetic oligonucleotides.<br />Synthetic oligonucleotides is used for invitro mutagenesis. In this method a small oligonucleotides primer containing the desired modification is first synthesized. It is then hybridized to the appropriate site and cloned gene and then the rest is replicated using DNA polymerase enzyme, so that the rest remains unaltered. This approach is actually used to modify the active site of the tyrosyl-tRNAsynthetase<br />
Synthesis of complete modified gene de novo<br />Complete gene in some cases have been chemically synthesized in the form of several oligomers (e.g. genes for insulin, somatostain and interferon), that are ligated in correct order to produce a complete gene. The sequence of the synthetic gene can be designed in a modular fashion to get the desired function.<br />
Computer<br />Modeling<br />Gene <br />construction<br />Protein <br />production<br />characterization<br />De novo design of proteins: The attempt to choose <br />an amino acid sequence that is unrelated to any <br />natural sequence, but will fold into a desired 3-D <br />structure with desired properties.<br />
Chemical modification of enzymes<br />The protein synthesized under the control of gene sequence in a cell undergo post-transitional modification. This leads to stability, structural integrity, altered solubility and viscosity of individual proteins.<br />for e.g. Enzyme-PEG conjugates. An enzyme L-asparaginase has antitumour properties but is toxic with a life time of less then 18hrs thus reducing its utility. <br />Continue……….<br />
L-asparginase can be modified by polyethene glycol derivatives to produce PEG-asparginase conjugates, which differ from the native enzyme in the following way (i) it retains only 52% of the catalytic activity of the native. (ii) it become resistant to proteolytic degradation. (iii) it doesn’t cause allergy.<br />
Achievements of protein engineering<br />A number of proteins are known now where efforts have been made to know the effects of site specific mutagenesis involving substitution of one or more amino acids.<br />Insulin- it consist of A and B chains are linked by C-peptide of 35 amino acids. It was shown that a sequence of 6 amino acids for c-peptide was adequate for the linking function.<br />Continue….<br />
cytochrome c – A phenylalanine residue has been identified to be non-essential for electron transfer but is involved in determining the reduction potential of the protein.<br />Trypsin- It could be redesigned to have altered substrate specificity.<br />
Acetylcholine receptor. This protein is involved in transport, of acetylcholine through. the membrane. Specific regions of this protein involved in acetylcholine binding and channel formation have been, identified.<br />Human beta interferon.<br />Removal of one of the three cysteine residues' I led to an improvement in stability of the enzyme.<br />
Conclusion<br />Substantial progress has been made in the of engineered protein by DNA technology . Observations from Genetic modification and Chemical modification , Regarded as redesigning nature rather than copying nature. Protein engineering and genetic engineering are the complementary fields to each other the successes in protein engineering will transform the way we make foods, drugs and chemicals.. Design of protein engineering using DNA technology remains challenging: Though some solutions have been found, few guarantees of success currently exist.<br />
References<br />Kuhlman, Brian; Dantas, Gautam; Ireton, Gregory C.; Varani, Gabriele; Stoddard, Barry L. & Baker, David (2003), "Design of a Novel Globular Protein Fold with Atomic-Level Accuracy", Science302 (5649): 1364–1368,<br />2. Looger, Loren L.; Dwyer, Mary A.; Smith, James J. & Hellinga, Homme W. (2003), "Computational design of receptor and sensor proteins with novel functions", Nature423 (6936): 185–190.<br />3. www.molecular-plant-biotechnology.info<br />