Oriented Covalent Immobilization of Engineered ZZ-Cys onto Maleimide-Sepharose: An Affinity Platform for IgG Purification

Bao, Ru-Meng; Yang, Hong-Ming; Yu, Chang-Mei; Tang, Jin‐Bao

doi:10.1007/s10337-016-3146-5

Cited by 5 publications

(1 citation statement)

References 31 publications

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“…In another study, elastinlike-protein (ELP) was fused to Z domains for the purification and recovery of antibodies, and the authors concluded that ELP-ZZ presented a higher binding affinity to IgG than ELP-Z [54]. In addition, it was shown very recently that the ZZ-domain can bind to two IgG molecules when used as an affinity ligand for antibody purification [55]. Regarding higher IgG concentrations, the ZZ-CBM3:IgG binding ratio was less than 1:1.…”

Section: Testing Of the Functionality Of Cbm-x Fusionsmentioning

confidence: 99%

Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

2021

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Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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Section: Testing Of the Functionality Of Cbm-x Fusionsmentioning

confidence: 99%