Final Report Summary - BIOIMPROVE (Lessons from the genome of Escherichia coli: understanding heterologous expression of eukaryotic internal membrane proteins in bacterial cells)
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.
The main goal of this project is to understand the biogenesis and heterologous expression of eukaryotic internal membrane proteins in Escherichia coli. Therefore, we 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 use 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 apply this together with systematic libraries (Keio collection) of recombined single-gene deletions of E. coli as host.
Some bacterial variants with improved properties for functional expression of the GPCR wild type rat neurotensin receptor 1 (rNTR1) were isolated. These clones from the Keio collection not only produce increased levels of functional rNTR1, but in addition have improved growth performance when are cultivated at low temperature (which favors the overexpression of GPCRs). Comparisons of these bacterial variants on the expression of other GPCRs with different features, demonstrate that the improved characteristics are more dramatic when less stable GPCRs were tested.
After several genetic and microbiology experiments, we were thenceforth able to demonstrate that, unexpectedly, the observed phenotypes where not due to the gene deletion denoted in the Keio collection. Only after obtaining the sequence of the whole genome of the selected Keio clone with the best performance (qseB) and the wild type E. coli strain, we identified a single nucleotide substitution on the rpoD gene that appears to be the one responsible for the observed phenotype. This essential gene encodes the sigma 70 subunit of the RNA polymerase, a primary sigma factor during exponential growth, which targets RNA polymerase to a wide range of promoters essentials for normal growth.
Using a site directed genome mutagenesis strategy, we then created a new E. coli strain having only this mutation in the sigma factor. Receptor expression experiments demonstrated that the better phenotype displayed by the Keio clone qseB is only due to this single point mutation in the rpoD gene of E. coli.
The mutation found in the sigma factor gene, may have a role in the specific interaction between RNA polymerase and promoters in transcriptional activation. Thus, it is possible that this single point mutation modified the mRNA levels in the bacteria. In this way, the synthesis of the GPCR rNTR1 can be altered in such a way that the toxic effects occurring during receptor expression are diminished, and therefore the better phenotype described above can be explain. We tried to test this hypothesis by using real-time qPCR technique, but after several trials realize that it will be difficult to choose a proper reference gene, as the mutation in the sigma factor can have a broad and general impact in the expression of many genes.
Therefore, we performed a whole transcriptome assay by obtaining the RNA-seq of the E. coli strains under study, in order to find differentially expressed genes in a global approach. This technique allows us to conclude that the level of rNTR1 gene expression is similar in both strains.
We also performed the whole transcriptome data set for the strains without the rNTR1 gene expressed, in order to understand better what happens at the gene expression level when the GPCR is expressed in E. coli. Although the data analysis is still undergoing, a first overview revealed the following scenario: several gene pathways that are up-regulated when the GPCR is overexpressed, are down-regulated in the mutant strain. The same applies in the other way: genes that are down-regulated when the GPCR is overexpressed, are up-regulated in the mutant strain compared to the wild type E. coli strain used. A deeper analysis of these data is currently in process.
Having this information that a modification on the sigma factor can elicit a positive impact in the expression of GPCR in E. coli, we are currently working on generate new bacterial host, with different genetic backgrounds.
The information that emerge from this project, provide a deeper understanding of the processes of heterologous expression of IMPs in bacteria. This allows us to generate bacterial hosts optimized for enhanced GPCR production.
Improving the (functional) amount of these membrane proteins is a major step forward to get valuable structural information of these pharmaceutical important targets, opening the doors for the discovery of new drugs for the clinical therapy associated with many diseases.
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.
The main goal of this project is to understand the biogenesis and heterologous expression of eukaryotic internal membrane proteins in Escherichia coli. Therefore, we 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 use 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 apply this together with systematic libraries (Keio collection) of recombined single-gene deletions of E. coli as host.
Some bacterial variants with improved properties for functional expression of the GPCR wild type rat neurotensin receptor 1 (rNTR1) were isolated. These clones from the Keio collection not only produce increased levels of functional rNTR1, but in addition have improved growth performance when are cultivated at low temperature (which favors the overexpression of GPCRs). Comparisons of these bacterial variants on the expression of other GPCRs with different features, demonstrate that the improved characteristics are more dramatic when less stable GPCRs were tested.
After several genetic and microbiology experiments, we were thenceforth able to demonstrate that, unexpectedly, the observed phenotypes where not due to the gene deletion denoted in the Keio collection. Only after obtaining the sequence of the whole genome of the selected Keio clone with the best performance (qseB) and the wild type E. coli strain, we identified a single nucleotide substitution on the rpoD gene that appears to be the one responsible for the observed phenotype. This essential gene encodes the sigma 70 subunit of the RNA polymerase, a primary sigma factor during exponential growth, which targets RNA polymerase to a wide range of promoters essentials for normal growth.
Using a site directed genome mutagenesis strategy, we then created a new E. coli strain having only this mutation in the sigma factor. Receptor expression experiments demonstrated that the better phenotype displayed by the Keio clone qseB is only due to this single point mutation in the rpoD gene of E. coli.
The mutation found in the sigma factor gene, may have a role in the specific interaction between RNA polymerase and promoters in transcriptional activation. Thus, it is possible that this single point mutation modified the mRNA levels in the bacteria. In this way, the synthesis of the GPCR rNTR1 can be altered in such a way that the toxic effects occurring during receptor expression are diminished, and therefore the better phenotype described above can be explain. We tried to test this hypothesis by using real-time qPCR technique, but after several trials realize that it will be difficult to choose a proper reference gene, as the mutation in the sigma factor can have a broad and general impact in the expression of many genes.
Therefore, we performed a whole transcriptome assay by obtaining the RNA-seq of the E. coli strains under study, in order to find differentially expressed genes in a global approach. This technique allows us to conclude that the level of rNTR1 gene expression is similar in both strains.
We also performed the whole transcriptome data set for the strains without the rNTR1 gene expressed, in order to understand better what happens at the gene expression level when the GPCR is expressed in E. coli. Although the data analysis is still undergoing, a first overview revealed the following scenario: several gene pathways that are up-regulated when the GPCR is overexpressed, are down-regulated in the mutant strain. The same applies in the other way: genes that are down-regulated when the GPCR is overexpressed, are up-regulated in the mutant strain compared to the wild type E. coli strain used. A deeper analysis of these data is currently in process.
Having this information that a modification on the sigma factor can elicit a positive impact in the expression of GPCR in E. coli, we are currently working on generate new bacterial host, with different genetic backgrounds.
The information that emerge from this project, provide a deeper understanding of the processes of heterologous expression of IMPs in bacteria. This allows us to generate bacterial hosts optimized for enhanced GPCR production.
Improving the (functional) amount of these membrane proteins is a major step forward to get valuable structural information of these pharmaceutical important targets, opening the doors for the discovery of new drugs for the clinical therapy associated with many diseases.