Gluten is the essential determinant of bread-malcing and other wheat flour-based end use products quality. It consists of two components: glutenins and gliadins. Glutenins are divided in two groups according to their electrophoretic mobility: high molecular weight subunits (HMW-GS) and low molecular weight subunits (LMW-GS). They are linked together by covalent disulfide bonding and form very large polymeric structures. HMW-GS (x- and y-types) are encoded by two closely linked genes at the Glu-1 loci (GluAl, GluBl and GluDl) that are located on the long arms of homologous chromosomes one. Bread wheat usually contains 3-5 HMW-GS arising from the expression of lAx, lBx or lBy, lDx and IDy. Studies on quality characteristics of wheat flour have shown that the HMW-GS composition strongly affects dough properties. Therefore, it is very important to determine the effect of individual polypeptides on dough functionality, particularly if testing unusual subunits that could be included in breeding programs for improving end product quality. Nevertheless, due to the number and amount of HMW-GS, purification of individual polypeptides is difficult because it is time consuming and very often the purified protein is contaminated with other accompanying polypeptides. This contamination prevents the accurate evaluation of functional properties of the individual polypeptides in dough. In order to avoid those drawbacks, recent testing developments have greatly contributed to properly study the functionality of individual polypeptides in dough. On one hand, the small scale testing system, using a 2 g Mixograph, extensograph and micro baking has facilitated the study of functional properties of small amounts of exogenous protein incorporated into different sample flours.1 On the other hand, in-vitro expression techniques allowed the expression of large amounts of protein in heterologous systems.2'3 The advantage of the latter is the production of large amounts of correctly folded polypeptides for functional studies. This paper mainly considers the expression, purification and functional studies of the smallest HMW-GS, subunit 12.4* present in Triticum tauschii.
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