Preparation Of Protein Molecular Standards Using Prokaryotic Expression System

Protein molecular weight standards is a common experiment reagent applied to electrophoresis and blotting technique, which is used to determine protein molecular weight and point the result of electrophoresis and membrane transferred. Protein molecular weight standards is the mixtures of highly purified different molecular weight proteins. There are three kinds of corresponding protein molecular weight standards such as unstained molecular weight protein standard,prestained protein molecular weight marker and prestained protein molecular weight marker for western blot. Efficient production of protein molecular standard is crucial to commercialization of protein standard. The aims of our research were to overexpress seven recombinant proteins with defined molecule weight using prokaryotic expression system and investigate the feasible usage as unstained molecular weight protein standards and easy-usage indicator for the western blot of similiar tagged protein. Furthermore,truncated non-transmembrane domains of cellulose synthase from above mentioned protein standard was used as antigen to immune rabbit, specific polyclonal antiserum was prepared. The results laid the detection basis for further research on the expression of Hv–CesA and cellulose synthesis in cell wall.The major research results were as follows:1.Designed a common used molecular weight standard series for overexpression,Such as 15kD,30kD,45kD,60kD,75kD,90kD and 120kD,and determined the source of coding genes.Coding regions with fewer rare codon were used as templates and expressed in prokaryotic system. Seven different gene fragments coding the pre-setted molecular weights were obtained by PCR and ligated into expression vector pET-28a(+) ,the fusion proteins meet the required molecular weight as well as the isoelectric points were ranging from four to nine. The built constructs were confirmed by PCR and double digestion, positive constructs were transformed into E.coli BL21 star(DE3) for overexpression.2.Expression conditions, such as induction profiles and IPTG concentration were optimized for seven proteins. The 15kD,30kD,45kD,60kD and 90kD proteins were mainly expressed in the form of inclusion body.75kD and 120kD were expressed in soluble state.Seven Overexpressed protein were purified by cobalt chelating chromatography. 3.The fused his-tag of purified proteins were detected using Gelcode His-tag Stain method, the results showed that every protein had recombinant tag.4.Overexpressed truncated non-transmembrane domains of cellulose synthase from the above recombinant proteins was further purified by gel cutting. The gel containing the corresponding band was used for polyclonal antibody preparation. Western Blotting result showed the raised antibody were specific and with high titer.The study described an efficient method for preparation of protein molecular standards used for SDS-PAGE assay and western blotting. The raised antibody against truncated Hv–CesA laid the basis for mechanism research of cellulose synthesis and cell wall development.