NMR Monitoring of Accumulation and Folding of 15N-Labeled Protein Overexpressed in Pichia pastoris
| Autor: | Marc-André Delsuc, Cécile Klein, Marie-Françloise Gautier, Guy Moulin, Frédéric De Lamotte, Catherine Blanchard, Hélène Boze |
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| Rok vydání: | 2001 |
| Předmět: |
Protein Folding
Ultrafiltration Pichia Structural genomics law.invention Pichia pastoris 03 medical and health sciences FLAG-tag law Combinatorial Chemistry Techniques Nuclear Magnetic Resonance Biomolecular Triticum Plant Proteins 030304 developmental biology Detection limit 0303 health sciences Nitrogen Isotopes biology 030302 biochemistry & molecular biology Antigens Plant biology.organism_classification Recombinant Proteins Yeast Molecular Weight Biochemistry Recombinant DNA Fermentation Protons Carrier Proteins Biotechnology |
| Zdroj: | Protein Expression and Purification. 22:318-324 |
| ISSN: | 1046-5928 |
| Popis: | Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native 15N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a 15N-labeled protein is estimated as 20 μM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method. |
| Databáze: | OpenAIRE |
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