Study On Protein Orientation Immobilization Based On Phosphopantetheinyl Modification

Multi-enzyme co-immobilizationis is a kind of technique for immobilizing proteins by introducing special fusion tags,which can avoid the direct participation of enzyme protein molecules in the reaction and maintain the conformation of enzyme active center.This technology is expected to overcome the technical difficulties of traditional technology and promote the large-scale application of immobilized enzyme,this has become a hot spot in the research of immobilized technology.On the basis of covalent binding method,in order to minimize the spatial structure of the immobilized proteins,some special chemical reactions and biological modifications will be used to realize the ordered and directional immobilization of proteins(enzymes).The possible effects of active sites provide theoretical basis for exploring new methods of protein(enzyme)specific orientation and covalent immobilization,and this would lay a foundation for the further popularization of immobilized proteins(enzymes).In this study,we use commercialized available sulfhydryl protein agarose which were coupled resin as a carrier,then we construct a fusion expression vector by using fluorescent protein as a pattern protein,and we obtain SRP-GFP or ACP-X protein(X represents GFP or mCherry)through the expression and purifing system of E.coli.After the sulfhydryl group was blocked by 3,4-dibromomaleimide,the recognition sites of SRP or ACP were modified by Sfp to mediate the covalent binding fixation of the target protein.A new system of protein immobilization and multi-enzyme co-fixation was established.In this experiment,we mainly studied the modification process and the dynamic process of immobilization of the target protein.The immobilization amount of different fusion proteins was determined by the determination of protein concentration and the analysis of fluorescence intensity.The results show that ACP,as a fusion label,can not only promote the soluble expression of the target protein effectively,but also realize the directional covalent immobilization of the protein.The saturated immobilized amount of ACP-GFP and ACP-mCherry is 8.67 mg/mL and 8.78 mg/mL,respectively.The results of common fixation showed that when they were added to the immobilized system at the ratio of 3:1,1:1 and 1:3,it was found that the immobilized proteins were immobilized at the ratio of 3:1,1:1 and 1:3 respectively.To sum up,the method system of protein covalent,directional immobilization and multi-enzyme co-fixation were established successfully,which provided the basis for the realization of multi-enzyme co-fixation industrial production.