NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.
Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-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 (15)N-labeled protein is estimated as 20 microM 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.
de Lamotte
F
F
Unité e Biochimie et Biologie Moléculaire des Céréales, INRA, Montpellier, France. lamotte@ensam.inra.fr
Boze
H
H
Blanchard
C
C
Klein
C
C
Moulin
G
G
Gautier
M F
MF
Delsuc
M A
MA
D052179:Antigens, Plant; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014908:Triticum / Q000737:chemistry / Q000235:genetics*
D052179:Antigens, Plant; D002352:Carrier Proteins; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D011522:Protons; D011994:Recombinant Proteins; C511920:lipid transfer proteins, plant