Fiol, Antonio Tarragona; (1992) Cloning, production and site-directed mutagenesis of bovine pancreatic ribonuclease A in Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

Text Cloning, production and site-directed mutagenesis of bovine pancreatic ribonuclease A in Escherichia coli.pdf Download (9MB)

Abstract

The coding sequence for bovine pancreatic ribonuclease A (RNase) precursor has been cloned and produced in E. coli using polymerase chain reaction (PCR) techniques. The use of the coding sequence for RNase precursor to produce mature ribonuclease has proved successful. Pre-ribonuclease is exported to the periplasmic space of E. coli and in the process the signal sequence is removed thus producing mature ribonuclease. Formation of the disulfide bridges in ribonuclease is facilitated by the oxidative environment of the periplasm and fully active protein is obtained. Clones containing DNA coding for mutant enzymes have been obtained using the technique of recombinant circle polymerase chain reaction (RCPCR). This work was carried out to test the applicability of RCPCR for the production of mutant ribonucleases, and the RCPCR technology is now validated for this purpose. The kinetic constants of these mutants have been investigated using dinucleotide substrates. The findings are discussed in terms of the interaction of active site residues with dinucleotide substrates.

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