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Design, Expression and Function Study of IgG Bindinrotein CBD-SPG

Author: MaJun
Tutor: JinJian
School: Jiangnan University
Course: Biochemistry and Molecular Biology
Keywords: Streptococcal Protein G Cellulose Binding Domain affinity chromatography
CLC: Q51
Type: Master's thesis
Year: 2013
Downloads: 14
Quote: 0
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Abstract


Streptococcal Protein G, a bacterial cell wall protein whose C terminal has the affinityfor immunoglobulin G (IgG). Cellulose Binding Domain of cellulase, which has a wedge-likestructure that is involved in cellulase adsorption without degradation of cellulose. In this study,we attempt to construct a fusion protein composed of CBD and SPG domain, which has boththe function of IgG binding and cellulose affinity. It is a novel tool in immunoassay andaffinity chromatography applied in purification of IgG.We amplified the coding sequence of CBDclosgene from Clostridium cellulovorans andCBDtrigene from Trichoderma viride as template, and then inserted them into thepET28a-SPG, a E.coli transfer vector. The cloned pET28a-CBDclos-SPG andpET28a-CBDtri-SPG plasmid was transformed into E.coli BL21(DE3) and screened bykanamycin to get a positive recombinant. Recombinant E.coli BL21(DE3) cells were culturedin LB broth containing kanamycin until the optical density reaching to1at30℃. Then cellswere induced to express CBDclos-SPG、 CBDtri-CBD by addition of Isopropylβ-D-Thiogalactoside. The40kDa and30kDa protein can be detected by this method. TheCBDclos-SPG、CBDtri-CBD purified using Sgmacell50column exhibited plenty of fusionprotein binding capacity. After elution, the purity was greater than95%as estimated fromthe protein gel. For the binding assays, the purified CBDclos-SPG、CBDtri-CBD was boundonto different matrix first to check the combining weight of CBDclos-SPG、CBDtri-CBD toseveral different materials, then IgG capacity was examined. The result showed that Avicelph101, the CBDclos-SPG binding capacity is11.61mg/g(w/w), while the capacity of IgG is25.73mg/g(w/w).In addition, our study has explored the application of CBDclos-SPG. CBDclos-SPG bindingcellulose is used to purify IgG in rabbit serum and antigen capture. The result showed thatCBDclos-SPG binding cellulose can be used to purify IgG in rabbit serum and capture fusionprotein IFNβ-HSA by using HSA antibody.In this thesis, we obtained bi-functional protein CBDclos-SPG、CBDtri-CBD by designing,constructing vectors and expressing them in E.coli. We showed that the fusion proteinCBD-SPG has dual binding capacities of both IgG and cellulose. It can be used as a novel toolin fields of immunoassay and affinity chromatography.

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