en
Master
English

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

ContributorsMiric, Maja
Master program titleMaitrise universitaire en pharmacie
Defense date2011
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.

eng
Keywords
  • RNase A
  • Expression
  • Purification
  • E. coli
  • Site-directed mutagenesis
Citation (ISO format)
MIRIC, Maja. Cloning, expression and purification of bovine pancreatic ribonuclease A (RNase A). 2011.
Main files (1)
Master thesis
accessLevelRestricted
Identifiers
  • PID : unige:16454
729views
6downloads

Technical informations

Creation06/16/2011 5:00:00 PM
First validation06/16/2011 5:00:00 PM
Update time03/14/2023 4:52:30 PM
Status update03/14/2023 4:52:30 PM
Last indexation01/29/2024 7:11:45 PM
All rights reserved by Archive ouverte UNIGE and the University of GenevaunigeBlack