BIOIMPROVE

Lessons from the genome of Escherichia coli: understanding heterologous expression of eukaryotic internal membrane proteins in bacterial cells

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'Eukaryotic cells express integral membrane proteins (IMP) that exchange substances with their environment or receive signals in the form of small molecules, peptides or proteins. Most human IMPs are inherently difficult to study, because of complications in obtaining adequate amounts of stable and functional proteins. G protein-coupled receptors (GPCRs) are the largest IMP family in the human genome and constitute the most important class of drug targets in pharmaceutical discovery. Escherichia coli is the most used host for heterologous protein expressions, although yields and quality for GPCRs are usually not sufficient for protein characterizations. Currently, our comprehension of processes that influence the biogenesis of IMPs in bacteria is limited. Therefore, the goal of this proposal is to study how E. coli genes could regulate the heterologous production of eukaryotic IMPs, using GPCRs as a model system. In order to achieve this, we will make use of a selection strategy developed in the host laboratory that allows detecting functional GPCR expressed on E. coli and enriching cells for their functional expression level. We will apply this together with systematic libraries of recombined single-gene deletions or of overexpressed genes of E. coli. The information that will potentially emerge will allow a deeper understanding of the processes of heterologous expression of IMPs in bacteria and will also generate bacterial hosts optimized for enhanced GPCR production. The general results arising from this project could ultimately provide valuable information for the discovery of new drugs for therapy. With this proposal, an experienced researcher from Argentina will undertake mobility and carry out a period of: transfer of knowledge, skills diversification and career development in Europe. This will allow create long-term collaborations and the enhancement of mutually-beneficial cooperative networks relationships between the EU and his home country.'

Introduzione (Teaser)

Drug design against cellular receptors necessitates the elucidation of their three-dimensional conformation. In this context, European researchers developed a method to maximise the yield of receptor expression in bacteria.

Descrizione progetto (Article)

Eukaryotic cells receive and process signals from their environment through integral membrane proteins (IMP). IMPs are notoriously difficult to study due to complications in obtaining adequate amounts of stable and functional proteins. G protein-coupled receptors (GPCRs), one of the largest IMP families, is also an important class of drug targets in pharmaceutical discovery.

Escherichia coli are widely used as hosts for heterologous protein expression but the efficacy and yield of GPCRs is very low and unsuitable for protein characterisation studies. The EU-funded BIOIMPROVE (Lessons from the genome of Escherichia coli: understanding heterologous expression of eukaryotic internal membrane proteins in bacterial cells) project aimed to address this limitation and investigate the processes that influence the biogenesis of IMPs in bacteria. The work focused on genes that regulate the heterologous production of eukaryotic IMPs.

Scientists employed a system based on the enrichment of cells expressing high levels of functional GPCRs and utilised bacterial strains mutated in various factors. Their experiments identified certain bacterial variants with enhanced GPCR expression. Genetic analysis of these variants showed a nucleotide substitution in the rpoD gene, which encodes the sigma 70 subunit of the RNA polymerase. During exponential growth, this factor targets the polymerase to genes that are required for normal growth.

The identified sigma mutant suggests that enhanced transcriptional activation may be the answer in diminishing the toxic effects incurred during receptor expression. Gene expression analysis revealed significant pathway alterations in rpoD mutants compared to wild-type bacteria.

BIOIMPROVE results provide a deeper understanding of the process of heterologous expression of IMPs in bacteria, a stepping stone for optimising GPCR production. This is of paramount importance for conducting structural studies and delineating the function of these pharmaceutical targets.