Expression of deuterium-isotope-labelled protein in the yeast pichia pastoris for NMR studies.
Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D2O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D2O medium with protiated methanol as carbon source, or in 95% D2O medium containing deuterated methanol. A high level of uniform Calpha deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [1H/15N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues. including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of 2H/13C/15N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.
Morgan
W D
WD
Division of Molecular Structure, National Institute for Medical Research, London, UK. wmorgan@nimr.mrc.ac.uk
Kragt
A
A
Feeney
J
J
D000595:Amino Acid Sequence; D000818:Animals; D017077:Culture Media, Conditioned; D003903:Deuterium; D017666:Deuterium Oxide / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D020066:Merozoite Surface Protein 1 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010963:Plasmodium falciparum; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D017077:Culture Media, Conditioned; D020066:Merozoite Surface Protein 1; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003903:Deuterium; D017666:Deuterium Oxide