Unfortunately, SDS-PAGE and Western blotting detection of the induced α-gliadin fusion proteins expressed in E. coli confirmed Pexidartinib datasheet that the high-level expression of α-gliadin in vitro was still difficult, although

the T7 promoter induced by IPTG was a suitable promoter for inducing the expression of α-gliadin genes in E. coli. Consequently, such potential contributions to gluten quality were not successfully identified by functional analysis in vitro. Fortunately, the functionality of a protein is determined largely by its three-dimensional structure, produced by folding secondary structures into one or several domains. Knowledge of the secondary structure of a protein may provide clues to its molecular function [34].

Generally, X-ray crystallography and nucleic magnetic resonance spectroscopy (NMR) are the two major experimental methods to determine protein structures accurately, but owing to their complexity, high cost, and time-consuming nature, progress on protein structure determination can be slow. As a result, over the last few years, computer-based automatic methods including GOR, PSIPRED, YASPIN and HNN have been developed for the rapid prediction, evaluation, and visualization of protein structures [34] and [35]. Of the most frequently used online software, PSIPRED is find more the most popular program and has several advantages over other programs including higher prediction accuracy, graphical and colored output of results, description of the confidence score values of each secondary structure element, and

the facility to download results in PDF format [34] and [36]. However, at present, the prediction of the secondary structures of α-gliadins is still very limited. Using PSIPRED version 2.6, Xie et al. [23] predicted the secondary structures of 19 full-ORF α-gliadins that they isolated from common wheat cultivars and Aegilops tauchii accessions and Bumetanide found that the numbers of α-helices and β-strands were not evenly distributed in the different proteins: a high content of β-strands and most of the α-helices and β-strands were found in the two unique domains, and in particular, more secondary structures were present in the C-terminal unique domain II. In addition, few or even no secondary structures were distributed in the N-terminal repetitive domain and glutamine repeat I. They accordingly inferred the C-terminal unique domain II to be the most important domain for the formation of intermolecular disulfide bonds with HMW and LMW glutenins. To ensure the accuracy and comparability of the results, the secondary structure of a total of 198 deduced typical α-gliadins, including the 22 genes cloned in this study, as well as the abovementioned 19 full-ORF genes, were predicted in the present study.