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Cloning, expression and purification of bovine pancreatic ribonuclease A (RNase A)

Denomination Maitrise universitaire en pharmacie
Defense Maîtrise : Univ. Genève, 2011
Abstract Bovine pancreatic ribonuclease A (RNase A) is a small basic protein with an isoelectric point of 8.46 and consisting of 124 amino acids (mature enzyme) that are stabilized by four disulfide bridges which made it difficult to apply protein engineering techniques due to problems in heterologous expression. In the past various expression systems have been established, e.g. E. coli, S. cerevisiae, or P. pastoris, have been established to address this issue. Amongst them, bacterial systems based on E. coli are preferred due to ease of use, low cost and fast growth and subsequent accumulation of significant cell mass. So far, such systems only produced RNase A as inactive form in inclusion bodies or low yields of soluble active enzyme. It is the aim of this project to design a new bacterial expression system that will allow the recombinant expression of soluble RNase A using modern bacterial strains and new expression plasmids that have been developed to produce disulfide containing proteins. Once the expression system has been achieved for the wild type enzyme, a series of amino acid mutations will be applied and the corresponding mutant enzymes expressed. All enzymes will be purified and analyzed regarding yield, purity, structure, activity and substrate/inhibitor binding.
Keywords RNase AExpressionPurificationE. coliSite-directed mutagenesis
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MIRIC, Maja. Cloning, expression and purification of bovine pancreatic ribonuclease A (RNase A). Université de Genève. Maîtrise, 2011. https://archive-ouverte.unige.ch/unige:16454

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Deposited on : 2011-06-28

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