Study On HMW-Gs(1Dx5,1Dy10) Fusion Expression In E.Coli

Studies show that high molecular weight glutenin subunits 1Dx5+1Dy10 (or sub5+sub10) controlled by two tightly linked genes on 1D chromosome may play an essential role in bread baking quality. Nowadays, It is one of important research fields to improve wheat flour processing quality by transformation of HMW-GS 1Dx5+1Dy10 genes with wheat cultivars. However no satisfying results have been obtained up to now because of the complexities of wheat gene expression and the knowledge shortage of roles of sub5 and sub10 played in bread baking processes. In order to study sub5 and sub10 structures and roles in bread baking further, 1Dx5, 1Dy10 genes are separately expressed in prokaryotic cell in current paper. The main results are as follows:Both the glutenin subunits 1Dx5 and 1Dy10 were cloned into the vector pRSETA, and then introduced into E.coli strain BL21(DE3)plysS, which will possess double resistances for antibiotics when the recombinant is transformed in the host. The gene was constructed under T₇ promoter control as a fusion protein, and the expressed products were easily distributed and purified with 6xHis Tag at its N-terminal. After induced of IPTG for 5 hours, the cells were collected and broken, and the expressions were extracted and examined by SDS-PAGE analysis. The different conditions, such as culture media, induced time and dosage of IPTG, were used to improve the target protein expression level. The optimal culture conditions were as follows: for glutenin subunits 1Dx5 in 2YT culture medium by 5 hours and 1.0mmol.L^-1 IPTG inducing and as for 1Dy10 in SOB culture medium by 5 hours and 1.0mmol.L^-1 IPTG inducing. SDS-PAGE analysis showed that contents of recombinant subunits 1Dx5 and IDylO were about 16.3% and 7.4% in total cell soluble proteins respectively.