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Abstract

Biochemistry

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Published: July 20th, 2021

DOI:

10.3791/62804

1Department of Molecular Biology and Genetics, Çanakkale Onsekiz Mart University, 2Uluova Dairy Farm, 3Department of Nutrition, University of Nevada, 4Department of Food Science and Technology, University of Nebraska, 5Evolve BioSystems Inc., 6Department of Biomedical Engineering, Karabuk University, 7Department of Chemistry, Hacettepe University

Abstract

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

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Keywords Recombinant Production

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