HIGH-LEVEL SECRETION OF A WHEAT LIPID TRANSFER PROTEIN IN PICHIA-PASTORIS

Plant nonspecific lipid transfer proteins are small basic proteins wit h eight cysteine residues, all engaged in disulfide bonds. The sequenc e encoding the wheat 9-kDa LTP was cloned into the secretion vector pY AM7SP8 giving rise to pYTdltp4.90. Production in shake-flasks and a fe rmenter led to the synthesis of two major species of LTP: a larger tha n expected species of 14 kDa and a species of 10 kDa, close to the exp ected size of wheat LTP. When production was carried out in a fermente r 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 homogene ity by ammonium sulfate precipitation, gel filtration, and anion-excha nge chromatography, with a recovery yield of 36%. However, the molecul ar mass of rLTP, determined by mass spectrometry, is 9996 Da, while it s naturally occurring counterpart has a molecular mass of 9607 Ha, Thi s discrepancy in size corresponds to a protein carrying three extra am ino acids (DKR) at its N-terminal end, and this was confirmed by seque ncing. In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein. These data indicate th at Pichia pastoris is an efficient system for production of large quan tities of soluble and biologically active rLTP for structure/function analysis. (C) 1998 Academic Press.