Abstract
BackgroundStudies on membrane proteins are often hampered by insufficient yields of the protein of interest. Several prokaryotic hosts have been tested for their applicability as production platform but still Escherichia coli by far is the one most commonly used. Nevertheless, it has been demonstrated that in some cases hosts other than E. coli are more appropriate for certain target proteins.ResultsHere we have developed an expression system for the heterologous production of membrane proteins using a single plasmid-based approach. The gammaproteobacterium Pseudomonas stutzeri was employed as a new production host. We investigated several basic microbiological features crucial for its handling in the laboratory. The organism belonging to bio-safety level one is a close relative of the human pathogen Pseudomonas aeruginosa. Pseudomonas stutzeri is comparable to E. coli regarding its growth and cultivation conditions. Several effective antibiotics were identified and a protocol for plasmid transformation was established. We present a workflow including cloning of the target proteins, small-scale screening for the best production conditions and finally large-scale production in the milligram range. The GFP folding assay was used for the rapid analysis of protein folding states. In summary, out of 36 heterologous target proteins, 20 were produced at high yields. Additionally, eight transporters derived from P. aeruginosa could be obtained with high yields. Upscaling of protein production and purification of a Gluconate:H+ Symporter (GntP) family transporter (STM2913) from Salmonella enterica to high purity was demonstrated.ConclusionsPseudomonas stutzeri is an alternative production host for membrane proteins with success rates comparable to E. coli. However, some proteins were produced with high yields in P. stutzeri but not in E. coli and vice versa. Therefore, P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins. Using the new pL2020 vector no additional cloning is required to test both hosts in parallel.
Highlights
Studies on membrane proteins are often hampered by insufficient yields of the protein of interest
P. stutzeri extends the spectrum of useful production hosts for membrane proteins and increases the success rate for highly produced proteins
Alternative prokaryotic hosts have been tested for their applicability for membrane protein production but only very few have been found to be successful in a similar extent as E. coli
Summary
Studies on membrane proteins are often hampered by insufficient yields of the protein of interest. Cells are surrounded by complex envelopes that control the exchange of metabolites, catabolites, energy and signals with the environment [1, 2] For this manifold requirements, they rely on membrane integrated proteins that differ in structure and function. To gain insight into the structure and function of membrane proteins, it is crucial to purify them in sufficient amounts and in a properly folded state [6] As they are usually not sufficiently abundant in the native cell membrane, numerous expression systems for the recombinant production of membrane proteins have been established. They differ in the host organism used, the transcriptional regulation or the post-translational modifications of the proteins [7, 8]. Due to their hydrophobic nature, studies on membrane proteins are challenging and in many cases already their production fails
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