Termoestabilización de proteínas de interés biotecnológico
Author
Rivera, Noé R.Entity
UAM. Departamento de Biología MolecularDate
2012-12-12Subjects
Bacterias termofílicas - Tesis doctorales; Ingeniería genética microbiana - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-12-2012Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
Thermostability is a desired property for many biotechnological
applications of enzymes because it allows an increase in their half-life at the
working temperatures and produces a concomitant increase in the stability
against organic solvents and detergents. Thermostable enzymes from
thermophilic microorganisms are well adapted to such conditions, but not are
always available or fulfill the role that the industry or specific applications
require, leading to the development of methods to select thermostable variants
from enzymes of mesophilic origin. Some of these methods rely on the deep
knowledge of the structure-function relationships of the enzyme to design
modifications that increase the thermostability. In other cases the methods of
“directed evolution” mimics nature by selecting for a specific property
(thermostability) among a high variety of random or semi-random mutants
generated by in vitro or in vivo procedures. In directed evolution, the success
of the selection depends on the capability to select over a huge amount of
variants, and require either robotic system for the analysis of individual
variants, or intermediate enrichment selection procedures to decrease the
burden of selection.
In this Thesis, we have applied the method of folding interference at
high temperature in a thermophilic host (Thermus thermophilus) to select
thermostable variants of two very different enzymes, a DNA polymerase form
bacteriophage φ29, and the esterase I from Pseudomonas fluorescens (PFEI).
The method is based on the expression in the thermophilic host of fusions
between the target protein (N-terminal) and a thermostable kanamycin
nucleotidyl transferase (C-terminal) as selectable reporter.
In the first part of the experimental work, we isolated T. thermophilus
deletion mutants defective in a major LonA-like protease, and checked their
suitability as selection host, leading us to conclude that this protease is highly
relevant to avoid toxicity of the fusions. In the second and third experimental
sections, we isolated “folding-reinforced” mutants of the exonuclease and
polymerase domains of the DNA polymerase of F29 and explained on
structural ground their effects on stability. In the final experimental section, we
went through the isolation of thermostable variants of the PFEI, and also were
able to explain most of the observed effects on structural models. Some of
the mutations obtained with both proteins are likely susceptible of patent
protection. General conclusions about what can be expected for the general
application of this method to isolate thermostable variants of any protein are
discussed.
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