Extraction and purification of low molecular weight glutenin subunits using size exclusion chromatography

Crude glutenin of commercial Indian wheat varieties was fractionated into high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) by employing size-exclusion chromatography (SEC). The SEC profile of glutenins obtained with different buffers were discriminated effectively with respect to the quality of the proteins eluted in each peak. The most efficient separation of LMW-GS was achieved using 3 M urea (pH 5.5) buffer under unreduced conditions. The chromatogram was segregated predominantly into three peaks with varied molecular weights as determined by SDS-PAGE. Peak I corresponded to a mixture of HMW-GS and LMW-GS (M_w 100-30 kDa). Peak II enclosed LMW-GS specifically with molecular weights in the range of 45-35 kDa. Lastly, a mixture of proteins associated with LMW-GS (M_w < 35 kDa) were eluted in peak III. SEC proved to be a valuable tool in purifying LMW-GS in a functionally active state.