The protein engineering is an important tool to produce superior enzymes and storage proteins. To do this, a protein engineer is required to prepare a computer aided protein model for a specific function and then prepare a synthetic gene that will produce this desired protein in a pre-decided and predictable manner. Using the technique of ‘site directed mutagenesis’, changes are made in genes at known sites leading to the modification of function in a predetermined manner. The ‘computational and graphical tools’ allow the display and exploration of three dimensional structures of proteins. These two techniques are very useful in deciding about the three dimensional structure of protein/drug needed for a specific purpose before using recombinant DNA technology for proteins or organic synthesis for drugs.
a) Preparation of a protein sample from an organism.
b) Characterization of the sample with a ligand such as enzyme-substrate, receptor-hormone or antigen-antibody interaction.
c) Analysis of the three dimensional (3-D) structure through NMR (nuclear magnetic resonance) and X-ray diffraction pattern of crystals.
d) Displaying of the 3-D structure with interactive computer graphics.
e) Processing of the information to select a novel design suitable to the required needs.
f) The desired DNA sequence which is expected to give the novel designed protein is then either synthesized or obtained by site directed mutagenesis of an available gene.
g) Introduction of the novel gene into a suitable expression system followed by it’s purification and biochemical characterization.
h) If biochemical characterization does not satisfy the earlier predicted structure, the cycle is repeated again.
Uses of protein engineering
The protein engineering is being used
a) to create superior enzymes with the capacity to catalyze the production of high value specific chemicals;
b) in the production of enzymes for large scale use in the chemical industry;
c) in the production of biological compounds including synthetic peptides, storage proteins and specific drugs that are superior to the natural ones.
There is an impressive list of proteins that have been altered using protein engineering technology. In ‘T4 Lysozyme’, a mutation of isoleucine to cystine leads to the formation of a disulphide bridge which gave thermal stability and a 200 fold increase in enzyme activity at a temperature of 670c. The removal of one of the three cysteine residues in the human beta interferon led to an improvement in stability of the enzyme. The enzyme ‘Trypsin’ could be redesigned to have altered substrate specificity. The substrate specificity of lactic protease (in E. coli.) has been shown to be dramatically modified by replacing active site methionine by alanine.
Protein engineering is also being used to create immunotoxins, which are the conjugates of cell binding antibody or antigens, covalently bound to a plant or a bacterial toxin. The procedure of fusion of genes using sequences coding for antibodies and toxic peptides is used to create these immunotoxins. When a patient is treated with the immunotoxins, the antibody or antigen helps in the recognition of the target cells to be killed and the toxin component helps in killing these cells. So immunotoxins are created in such a way that they have two parts- a) a toxin polypeptide or a part of it having toxin activity, called A chain, and b) a cell binding recognition polypeptide or antibody or a part of it having binding site, called B chain. E.g. Ricin, a plant toxin has actually been used as a immunotoxin for a study of it’s effect on mouse tumour cells.
Drug designing is another upcoming area in biotechnology. Depending on the mode of action used by the drugs, the drug designing can be modified by blocking the enzyme activity. E.g. Trimethoprim (TMP) is a clinically important antimicrobial drug which inhibits the enzyme dihydrofolate reductase (dHFR) in bacteria and therefore, is used to treat urinary tract infections. However in high concentrations it starts attacking human dHFR thus becoming harmful. Efforts have been made to synthesize TMP, which will have a rigid three dimensional structure in association with bacterial enzyme dHFR, so that it may not be able to attack human dHFR.
Another example is of enzyme Renin. Inhibitors of enzyme ‘renin’ are also being actively modeled. The enzyme catalyses, the first in a series of reactions that lead to elevated blood pressure. The efforts are going on to design nonpeptide inhibitors that mimic the intermediate products in the reaction of renin with it’s substrate and thereby stops the functioning of renin. These inhibitors will help in treating the hypertension.
Drugs are also being produced by studying the receptor molecules associated with a particular disease and then finding chemicals which can block these receptors. One of the examples is the drug ‘Propranolol’ which is used to treat heart diseases and hypertension. The two hormones, ‘norepinephrine’ and ‘epinephrine’ controls the contraction and relaxation of cardiac muscles. These hormones act through the two receptors alpha (œ ) and beta ( ß). The drug propranolol blocks the receptor beta (ß ) and thereby inhibits the activity of hormones which act through a receptor molecule. This relaxes the heart muscle. This drug is being used to treat heart diseases, angina pectoris and high blood pressure.
Another drug, ‘Cimetidine’ is an antiulcer medicine given to treat ulcers. Ulcers are caused from acid production due to histamine release in the stomach. The drug ‘Cimetidine’ blocks the histamine receptors which prevents the acid production due to no availability of histamine. This leads to the healing of ulcers.
The detailed studies of antimetabolites or inhibitors of DNA synthesis has led to the development of drugs for treating ‘cancer’, ‘gout’, ‘malaria’ and some viral infections like Herpes. Drugs like ‘6-mercaptopurine’ and ‘thioguanine’ by inhibiting the DNA synthesis and the cell division, are very effective in cancer chemotherapy. Based on the same principle of inhibition of nucleic acid synthesis, the drugs like ‘pyramethamine’ for malaria, ‘trimethoprim’ for bacterial infection, and ‘acyclovir’ for herpes virus infection etc were also developed.
The area of research in drug designing and development of immunotoxins is receiving lots of emphasis as it may be the answer for all the future problems associated with the diseases related to new viruses and resistant bacterias. It may also give a breakthrough for treating diseases like AIDS and cancer which continue to challenge and threaten the human population.
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