High-level secretion of a wheat lipid transfer protein in Pichia pastoris.
Plant nonspecific lipid transfer proteins are small basic proteins with eight cysteine residues, all engaged in disulfide bonds. The sequence encoding the wheat 9-kDa LTP was cloned into the secretion vector pYAM7SP8 giving rise to pYTdltp4.90. Production in shake-flasks and a fermentor led to the synthesis of two major species of LTP: a larger than expected species of 14 kDa and a species of 10 kDa, close to the expected size of wheat LTP. When production was carried out in a fermentor with regulation of pH, oxygen level, and feed rate of carbon source, the 10-kDa species was the main protein at the end-point of culture. The recombinant wheat LTP (rLTP), secreted at a level of 720 mg/liter into the culture medium, is soluble. The rLTP was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography, with a recovery yield of 36%. However, the molecular mass of rLTP, determined by mass spectrometry, is 9996 Da, while its naturally occurring counterpart has a molecular mass of 9607 Da. This discrepancy in size corresponds to a protein carrying three extra amino acids (DKR) at its N-terminal end, and this was confirmed by sequencing. In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein. These data indicate that Pichia pastoris is an efficient system for production of large quantities of soluble and biologically active rLTP for structure/function analysis.
Klein
C
C
Unité de Biochimie et Biologie Moléculaire des Céréales, INRA, Montpellier, France.
de Lamotte-Guéry
F
F
Gautier
F
F
Moulin
G
G
Boze
H
H
Joudrier
P
P
Gautier
M F
MF
D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014908:Triticum / Q000378:metabolism*
D052179:Antigens, Plant; D002352:Carrier Proteins; D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; C511920:lipid transfer proteins, plant