Cloning of a wheat 15‐kDa grain softness protein (GSP): GSP is a mixture of puroindoline‐like polypeptides

The wheat starch 15‐kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15‐kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti‐GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15‐kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15‐kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half‐way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16‐kDa avenin and to wheat puroindoline (a lipid‐binding 15‐kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid‐binding properties of GSP polypeptides may influence grain softness.