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Membrane proteins are of fundamental importance for all living organisms. They represent up to 30% of all genes in the genomes of prokaryotes and eukaryotes. The malfunction of membrane proteins can have fatal consequences for the cell making them an attractive drug target. About 40% of all membrane proteins are involved in the transport of substrates across the membrane. Thereby they can passively facilitate the permeation of their substrates or actively transport one or several substrates in or out of the cell. In the latter case the energy is derived from the consumption of ATP (pumps) or from an electrochemical ion gradient (secondary active transport). The different transport mechanisms are based on structural differences of the transporter classes. It is therefore crucial to gain structural insight into the transport mechanism of different transport proteins. This is often hampered by low yields, misfolding and aggregation during the production and purification process.
In this work, members of the Auxin Efflux Carrier (AEC) family of secondary active transporters were selected from several thermophilic bacteria and archaea and tested for their production in E. coli. Additionally, the E. coli AEC transporter yfdV was selected and tested. The production of members of this family was generally difficult and most proteins were not produced under the conditions tested during this work. However, the E. coli homologue yfdV could be produced in a homologous system. E. coli LMG194 was used to produce the proteins from a pBAD vector utilizing the PBAD promoter. The production could be optimized to increase the yield of purified protein. The protein could be solubilized with different detergents but was found to be instable. By size exclusion chromatography the stability of the purified protein was compared in different buffers and further optimized. Still, the yield and also the stability were unsatisfactory. Therefore, the structural and functional characterization of these proteins was not further pursued.
The production of AEC transporters and also of many other families of membrane proteins repeatedly failed in in the most commonly used production host E. coli. Even though much effort has been made to optimize E. coli for this purpose, alternative strategies are required to make difficult to produce target proteins accessible for functional and structural studies. In this regard, the use of alternative production hosts has proven to be a promising strategy.
The gram-negative bacterium Pseudomonas stutzeri was used in our laboratory to investigate the terminal oxidases of the respiratory chain in the past years. The recombinant production of cbb3 oxidases was established and P. stutzeri therefore was tested as a potential new production host for membrane proteins.
The newly constructed expression vector pL2020 is functional in E. coli and P. stutzeri and presumably in most if not all gram-negative bacteria. It utilizes the PBAD promoter allowing tight control of expression levels from this vector. The production of a set of 36 secondary active transporters was tested in P. stutzeri and the results were compared to results obtained with E. coli in a previous study. In P. stutzeri, 20 of the target proteins were scored with high and six with low level production, respectively. In E. coli only 16 proteins were produced at high levels. However, 24 target proteins could be produced at high levels in any of the two hosts and only four were not produced at all. P. stutzeri is therefore a useful addition to E. coli. Moreover, eight membrane proteins derived from P. aeruginosa were also tested in P. stutzeri and could be produced at high levels. The P. stutzeri system provides a robust production platform for proteins derived from “quasi homologous” sources. A subset of the target proteins was fused to the N-terminus of GFP to analyze their folding in E. coli and P. stutzeri. For most of the tested proteins the portion and also the total yield of the folded protein species was higher in P. stutzeri. Five members of the Gluconate:H+ Symporter (GntP) family were included in the test set of 36 secondary active transporters. While they could not be produced in E. coli in sufficient amounts, three of the proteins were obtained at high levels in P. stutzeri. The Salmonella enterica protein STM2913 was chosen for large-scale production and purification trials. The protein could be purified with high purity and a yield of >0.1 mg/L of bacterial culture. The stability and homogeneity of the protein was demonstrated by size exclusion chromatography. Dynamic light scattering was used to evaluate the influence of different detergents on the stability. The detergent 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside (LM-NG) was chosen for crystallization trials and yielded crystals during Lipidic Cubic phase Crystallization.
In summary, P. stutzeri was established as a new useful production host in addition to
E. coli. The large-scale production, purification and initial crystallization of STM2913
was achieved with this new system but not with E. coli before.
