Solubility and manipulation of disulfides in puroindoline-b: Recombinant puroindoline-b shows antifungal activity

Abstract

Wheat (Triticum aestivum) kernel texture (hardness) is the most important determinant of milling and end-product quality. Recent data indicate that the only difference between soft and hard textured wheat is a single amino acid mutation in one protein, puroindoline-b (PIN-b). A rare tryptophan-rich domain in this protein consists of five tryptophan residues among a stretch of seven amino acid residues. To understand how this single mutation makes hard wheat possible, thus enabling bread making, it is crucial to have a high-resolution three-dimensional structure of this protein. The prerequisite for structural elucidation of any protein is the high-quality sample preparation. In this thesis PIN-b from a diploid wheat (Triticum monococcum ) was chosen as the model system because its grain is soft and it has the simplest genome of all wheats. The coding sequence of PIN-b was amplified from the diploid wheat using PIN-b specific primers. It was cloned into a protein expression vector. PIN-b was expressed as a protein behind the thioredoxin (TRX-a) tag. The TRX-a-PIN-b fusion protein was purified using nickel chelating chromatography. The immunological identity of the fusion protein was confirmed by Western blot. The PIN-b was released from the fusion protein by enterokinase proteolysis and purified using ion exchange chromatography. After glutathione treatment to facilitate full formation of the potential five disulfide bonds, PIN-b demonstrated higher fungicidal activity when compared to the non-treated PIN-b.