Protein Science & Engineering

Nearly all life processes depend on the functioning of proteins; long polymers of building blocks known as amino acids which are joined together in sequences specified by the DNA (gene) responsible for overseeing their synthesis in the cell. Each chain of amino acids folds into a three dimensional structure that is usually, exquisitely adapted to the performance of a specific, required 'molecular-recognition' function (e.g., binding/transport/catalysis). Much of modern biotechnology, i.e. the technology that allows researchers to manipulate life processes, hinges on mankind's insights into (and newfound ability to tinker with) the synthesis, folding and function of proteins. Such tinkering is usually effected either through chemical modification, or through the manipulation of the encoding gene. Since the relationship between a protein's amino acid sequence (i.e., information available from sequencing of DNA) and three- dimensional structure (i.e., shape ultimately responsible for performance of function) has not yet been fully worked out, fundamental research is required to go hand-in-hand with applied research, towards the development of any novel protein-based technology.

The protein science and engineering group at IMTECH combines on overt set of interests of a fundamental nature with an equally overt leaning towards the finding of applications. Two of the projects are in the broad areas of structure determination (X-ray crystallography) and molecular modelling, with emphasis on proteins involved in signal transduction and key aspects of the metabolism of pathogenic microorganisms. Two others are in the area of protein structural biochemistry who utilize a combination of wet biochemistry, separation and analytical methods, spectroscopy, and recombinant DNA technology, to deal with a variety of fundamental and applied issues relating to enzyme function, protein-protein interactions, protein folding, mis- folding, aggregation, sequence-structure relationships et cetera.

Between them, this group covers nearly the whole gamut of scientific and technical perspectives relating to proteins. In terms of applications, work is currently in progress in several areas e.g.

(i) thrombolytic protein agents that are useful in the treatment of infarctions,
(ii) inhibition of protein misfolding/aggregation, and enhancement of folding in vitro, etc.

The administration of "clot buster" drugs like streptokinase, TPA, urokinase etc can reduce loss of life by as much as 40 percent in case of myocardial infarction (heart attack), as well as other circulatory disorders. These agents work through the activation of plasmin in the circulatory system from its zymogen, plasminogen. Plasmin is a blood enzyme that proteolytically degrades fibrin, the principal component of blood clots to soluble products, thereby helping to restore blood supply to the afflicted tissue.

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• Development of bench-scale technology for the production and purification of therapeutic grade Natural Streptokinase :
  The Institute has been able to develop a bench scale technology for the production and purification of therapeutic grade natural streptokinase, a blood clot dissolving drug which is much needed for the treatment of myocardial infarction and other disorders. At present streptokinase is not manufactured in India and is wholly imported. Therefore, the development of this indigenous, cost effective technology for large scale purification of this drug by biotechnological means is an appreciable achievement. The Institute has successfully transferred this know-how to a leading drug company which has commercially launched this product in Indian markets under the brand name STPase.
  
  
• Development of Clot-Specific Streptokinase :
  It has been observed that in 1% cases clinically effective dose levels of thrombolytic drug like SK often result in systemic plasminogen activation as an unwanted side-effect, leading to the proteolytic degradation of clotting factors, including fibrinogen, and consequent hemorrhagic complications in a significant proportion of patients. IMTECH scientists have designed and expressed novel, second-generation SK molecules through rDNA technology that possess a strong fibrin affinity as well as an initially delayed rate of PG activation ("Time-delayed activation"). The simultaneous presence of these two properties should result in considerably reducing and/or minimizing the generalized PG activation observed following the administration of these modified thrombolytic derivatives in vivo.

  An enhanced fibrin affinity engineered into SK superimposed onto the property of a delayed PG activation rate is expected to be of great advantage clinically since these derivatives could circulate in an inactive state for several minutes soon after administration, allowing them to "home" onto the pathological clot by virtue of their strong fibrin affinity. Thus, after administration in vivo, clot lysis will occur predominantly in the immediate vicinity of the clot, leading to a continued and more efficient fibrinolysis in the absence of a generalized proteolysis in the circulation. Various clot-specific, delayed-action derivatives of SK have been designed and constructed that activate PG with an initial hiatus of several minutes duration.




• Structure-function relationship in Staphylokinase and generation of its novel genetically engineered derivatives with improved thrombolytic characteristics :
  Streptokinase (SK) and Staphylokinase (SAK) are two fibrinolytic agents of bacterial origin. Thrombolytic potential of SAK remained unexplored for long time due to unavailability of large amount of pure protein because of very low level of SAK elaborated by the producer strain. However, recent studies using recombinant DNA approach has clearly indicated that SAK may be developed as an alternate thrombolytic agent particularaly due to its smaller size, lower antigenicity and relatively greater fibrin-specificity. Recent clinical trials on recombninant SAK may be developed as an alternate thrombolytic agent particularly due to its smaller size, lower antigenicity and relatively greater fibrin-specificity. This project aims to understand the structure function aspects of SAK and develop new variants of SAK by site- specific mutagenesis, DNA shuffling or by generating chimeric variants carrying functional units of both SAK and SK in order to generate new molecule which may display improved functional properties e.g. higher functional activity, protein stability, and specificity towards fibrin and plasmin-inhibitors.
  
Besides the above mentioned the following projects which are designed to address questions of more basic nature in the area of protein science are also being carried out:
• Inhibition of protein misfolding / aggregation and enhancement of folding in vitro.


• Protein interactions in signalling proteins: Structure of SH3 domain and its target proteins.


• An analysis of protein structure to elicit the principles for knowledge based protein modelling.

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