Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation

Publication date: Available online 10 February 2019Source: Colloids and Surfaces B: BiointerfacesAuthor(s): Jing-Hong Wang, Ming-Ze Tang, Xiao-Tian Yu, Chong-Mei Xu, Hong-Ming Yang, Jin-Bao TangAbstractEnterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)4-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EKLC on amine-modified magnetic nanoparticles (NH2-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EKLC@NH2-MNP biocatalyst. Upon the site-specific immobilization, approximately 90 % EKLC enzymatic activity was retained, and the biocatalyst exhibited more than 85 % of initial enzymatic activity regardless of storage or reusable stability over a month. The EKLC@NH2-MNP biocatalyst was further applied to remove the His tag-(Asp)4-Lys fusion partner from the His tag-(Asp)4-Lys-(GLP-1)3 substrate fusion protein, result suggested the EKLC@NH2-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P >  0.05). Supported by the unique properties of MNPs, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein c...
Source: Colloids and Surfaces B: Biointerfaces - Category: Biochemistry Source Type: research