Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production.
The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 μg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.
Institute for Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany. christian.berger@medizin.uni-leipzig.de
Christian
C
Berger
Cindy
C
Montag
Sandra
S
Berndt
Daniel
D
Huster
D003470:Culture Media; D046508:Culture Techniques; D004926:Escherichia coli / Q000254:growth & development*; D005285:Fermentation; D006801:Humans; D007553:Isotope Labeling; D019906:Nuclear Magnetic Resonance, Biomolecular; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D003470:Culture Media; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; C095889:neuropeptide Y2 receptor