Desarrollo de nuevas herramientas y protocolos de selección de proteínas termoestables en "Thermus thermophilus"

Abstract

The genus Thermus has became one of the most used models for bacterial thermophiles because of its easy handling in the laboratory, its high cultures yields under aerobic conditions, and its natural competence. These properties, and the ability of its thermophilic enzymes and supramolecular complexes to crystallize at room temperatures, are on the basis of the most relevant acomplishment in recent years in the structural biology filed, with paradigmatic examples as relevant as the high resolution structures of 70S ribosome, the bacterial RNA polymerase and the respiratory complex I. To stablish a clear link between structure and function a well developed resource of genetic tools are required, and this Thesis includes some of the most recent attemps to provide new tools for the genetic analysis of Thermus. At the time when this work started, a single antibiotic resistance (to kanamycin) could be used as selectable marker at high temperatures, and any further manipulation of the strains would require the deletion of the marker in order to be used again on the same strain. To overcome this, we attempted to use alternative gene selection markers. For this, we followed two strategies. One one hand, we took advantage of the development in 2005 by a japanese group (Nakamura, 2005) of a thermostable form of a hygromycin B phosphotransferase to develop new plasmids which are compatible with the resistance to kanamycin, thus allowing protein expression and complementation assays in kanamyn resistant background. The new hygromycin resistant plasmids are described in the first chapter of the Thesis.

On the other hand, in the second chapter we describe the development of a new marker based on a spontaneous allele of the rpsL gene coding for a mutant S12 protein that provides not only resistance but dependence from Streptomomycin. Thus, such marker, allowed its use not just for positive selection to develop new plasmids, but also to carry out directed deletions of genes by using it for negative selection. We illustrate the use of this new marker by the expression of genes in T. thermophilus, by the complementation of mutants in kanamycin-resistant backgrounds, and through the isolation of a deletion mutant in a straight-forward strategy. The third chapter is devoted to describe the use of a mutant form of the Green Fluorescent Protein of Aquoeroa victoria as a localization marker in T. thermophilus. We demosntrate that a mutant form of this protein called superfolder GFP can be expressed and processed in T. thermophilus at 70º C to render a fully fluorescent protein which utility is demonstarted by expressing it as C-terminal fusions to as cytoplasmic and a periplasmic protein. Noteworthly, we demonstrate that this sGFP can be secreted to the periplasm at 70º C in a fully active and folded protein. In the last chapter of the Thesis we developed a new and general method for the stabilization of proteins through a protocol of selection in Thermus thermophilus, in collaboration with a biotechnology company specialized in protein evolution. The method is based on the interference that a unstable Nterminal part of a protein has on the folding of its C-terminal domain, in such a way that we developed plasmids from wich protein fusions between a target and a reporter coding for a thermostable resistance marker could be expressed in T. thermophilus at thermophilic temperatures. By this strategy we could demonstrate this principle and select thermostable forms of a the human gamma interferon, a protein that doesn't have any enzymatic activity, in a very rapid and efficient protocol. The general application of this method to other proteins and enzymes could also be demonstrated further in the biotech company.