Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Final Activity Report Summary - PROTENG (Engineering Proteases)

The project aimed at the fishing and cloning of known and novel proteases by appropriate technology including the company's proprietary cluster screening technology. Furthermore, it should help to build up the company's technology platform for the construction of libraries, the screening for interesting enzymes, their biochemical analysis, and the development of effective production and purification systems. We were able to build and express protease libraries in prokaryotic and eukaryotic systems, to establish suitable screening systems using fluorescence markers and to isolate interesting proteases as well from environmental sources, e.g. novel types of the alkali subtilisin-type protease and the neutral protease from Bacilli species, as from variant libraries of already well-characterised enzymes, e.g. human and bovine chymosin and human caspase-3.

The project helped to optimise the methods for the production of variant libraries on the DNA level, their transformation into suitable expression hosts at high titers, cultivation and gene expression in miniaturised systems, semi-automated purification of enzymes in the micro-scale, establishment of enzyme activity assays, especially for the fluorescent detection, isolation of promising genes based on the cluster-screening technology, production and purification in the laboratory and pilot scale. The project especially focused on establishing prokaryotic and eukaryotic expression systems for the production of proteases for industrial and therapeutic use.

We established the use of Escherichia coli for cytosolic and periplasmatic expression, and of Bacillus subtilis, Bacillus amyloliquefaciens, Pichia pastoris and Kluyveromyces lactis for secretion of enzymes. A major challenge in the project was the toxicity of proteases towards the expression hosts. This problem was observed with both, prokaryotic and eukaryotic systems. A lot of effort was spent into the optimisation of the expression systems to overcome this problem. The problem could be partially solved by the introduction of new expression hosts, e.g. of P. pastoris or B. subtilis, and by the construction of vector/host systems allowing for tighter control of induction of expression and improved secretion of the products.

Much effort was put into the establishment of a screening system for variant libraries of coagulation factors. Coagulation factors would be an optimal starting point for the development of proteolytic drugs to be introduced into the blood stream, e.g. in the therapy of cancer or atherosclerosis. We were able to produce active enzyme in the E. coli periplasma; however we were not successful in establishing a stable expression system, which would have been a prerequisite for the establishment of a variant library.

A lot of work was also invested in the optimisation of the screening systems, mainly in the production of eucaryotic libraries, which were large enough for screening a sufficient amount of different enzymes to find the desirable characteristics in terms of activity and stability. Eukaryotic hosts, such as P. pastoris, are of interest for enzymes, which cannot be produced in an active form in prokaryotic hosts. Mainly the transformation of eukaryotic hosts at rates sufficient to cover a whole library proved difficult, but also the cultivation in clusters suitable for screening proved. In the meantime, problems could partially be overcome by establishing methods for the transformation at reproducible, medium-high rates, and methods for cultivation of the library host. The methods established have already proven useful in enzyme screening. A major goal was also the production of proteases at a medium-to-large scale. We were able to produce novel engineered variants of caspase-3, bovine and camel chymosin and coagulation factor Xa at a large scale and establish appropriate purification protocols to achieve the required product quality.

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