English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

Pseudomonas stutzeri as an alternative host for membrane proteins

MPS-Authors
/persons/resource/persons215086

Sommer,  Manuel
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137954

Xie,  Hao       
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

/persons/resource/persons137800

Michel,  Hartmut       
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

External Resource
No external resources are shared
Fulltext (restricted access)
There are currently no full texts shared for your IP range.
Fulltext (public)
There are no public fulltexts stored in PuRe
Supplementary Material (public)
There is no public supplementary material available
Citation

Sommer, M., Xie, H., & Michel, H. (2017). Pseudomonas stutzeri as an alternative host for membrane proteins. Microbial Cell Factories, 16: 157. doi:10.1186/s12934-017-0771-0.


Cite as: https://hdl.handle.net/21.11116/0000-0001-27F3-1
Abstract
Background:
Studies 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.

Results:
Here 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.

Conclusions:
Pseudomonas 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.