Study On The Aggregation Behaviors Of Recommbinant N-terminal Domain Of High Molecular Weight Glutenin Subunit 1Dx5

Glutenin form gluten networks by the intermolecular disulfide bond, and decide the processing properties of the flour. High molecular weight glutenin subunits(HMW-GS) are the molecular basis for the backbone structure of glutenin aggregates, and their N-terminal domains are the key domain that influences the dough strength. 1Dx5 is a kind of HMW-GS with high quality. In this study, highly purified and soluble recombinant 1Dx5-N was prepared by genetic recombination and affinity chromatography technique. The aggregation behavior of the proteins in solution was investigated by electrophoresises, spectrometries and electron microscope techologies. The main results were followed as below:(1) After 1Dx5-N gene was cloned, the recombinant expression vector was constructed and then transferred into E.coli to produce the soluble recombinant proteins. After optimizing prokaryotic expression conditions, the optimum expression conditions were determined that the concentration of IPTG was 0.5 mmol/L, the induction temperature was 16?C and the induction time was 8 h. The fusion protein with high purity was obtained with metal chelating affinity chromatography column.(2) 1Dx5-N mainly formed large aggregates in aqueous solution. The fusion tag had no effects on the structure and aggregation behavior of 1Dx5-N. Furthmore, the effects of sodium dodecyl sulfate(SDS), dithiothreitol(DTT) on the aggregation behavior of 1Dx5-N were investigated. Although intermolecular disulfide bonds were formed, the hydrophobic interaction was the main driving force for the formation of 1Dx5 aggregates. Moreover, the hydrophobic interaction and disulfide bond were further strengthened with the increase of storage period.(3) The 1Dx5-N solution structure and aggregation were determined at different p H. 1Dx5-N formed large aggregates with particle size being greater than 100 nm in neutral p H condition, but the particle size was significantly reduced in acidic condition or in alkali condition. In addition, the secondary structure of 1Dx5-N was more ordered in acidic condition, but changes into disorder in alkali condition. Futhermore, the hydrophobic interaction between protein molecules was much stronger at p H 7.0 than that in acidic condition or in alkali condition with the storage time increasing, which facilitated the formation of the intermolecular disulfide bonds.(4) The effect of the concentration of Na Cl on the aggregation behavior of 1Dx5-N was studied. With the concentration of Na Cl increasing, 1Dx5-N was induced to form more aggregate particles. The electrostatic repulsion between protein molecules was considerably weakened with the addition of salt, which favored the growth of aggragate particles, the enhancement of hydrophobic forces and the formation of intermolecular disulfide bond.