Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Summary

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

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.

Introduction

Glycosylation is a very crucial post-translational modification observed in proteins. Approximately more than 50% of proteins are found in their glycosylated forms in eukaryotes. N- and O-glycosylation are the two major types of glycosylation^1,2. O-linked glycans (O-glycans) are covalently attached to proteins via N-acetylgalactosamine to the hydroxyl group of a serine (Ser) or threonine (Thr) amino acid residues. N-linked glycans (N-glycans) are complex oligosaccharides, which are covalently attached to asparagine (Asn) amino acid residue of the p....

Protocol

1. Molecular cloning of Bifidobacterial genes

1. PCR amplification of targeted genes by three vector primer sets (N-His, C-His, and N-His SUMO)
   1. Make 100 µM stock primer (oligomers) solutions by adding sterile water in the amounts determined by the company. Prepare 10 µM new stocks from these stocks to be used in PCR amplification of the target genes.
   2. Prepare the PCR mixture (total volume 50 µL) with 25 µL of master mix, 1 µL of forward and reverse primer at 0.2 µM, 21 µL of DNase/RNase-free distilled water and 2 µL of template DNA (bacterial cells) in the PCR tubes. Gently stir th....

Results

Glycosyl hydrolase member enzymes selected from different origins were targeted in this study. It was assumed that the co-application of different enzymes with different structures could provide a better glycan release since they are evolved to be active in different glycoproteins. The list of target genes and their origin is listed in Table 1. Bacterial strains were obtained from Belgium Co-ordinated Collections of Micro-organisms. Primer sets were designed based on the manufacturer's guidelines (

Discussion

The in vivo recombinational cloning strategy used for the molecular cloning of the target genes provides fast and reliable results compared to other traditional cloning protocols. Even though there are many convenient methods for molecular cloning, the method described in this article has more advantages. In vivo cloning system, unlike other cloning systems, does not need any enzymatic treatment or purification of the PCR products. Also, there is no limitation related to sequence junctions or the requir.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EconoTaq PLUS 2X Master Mix	Lucigen	30035-1	Amplification of target genes (PCR)
DNase/RNase-free distilled water	Invitrogen	10977035	Amplification of target genes (PCR)
Safe-Red Loading Dye	abm	G108-R	DNA gel electrophoresis
1 kb Plus DNA Ladder	GoldBio	D011-500	DNA gel electrophoresis
Qubit protein assay kit	Invitrogen	Q33211	Measurement of DNA concentration
LB Broth, Miller (Luria-Bertani)	amresco	J106-2KG	Bacterial culture media
Agarose	Invitrogen	16500-500	Bacterial culture mediaet al.
Kanamycin Monosulfate	GoldBio	K-120-5	Antibiotic in bacterial culture media
Expresso Rhamnose Cloning and Expression System Kit, N-His	Lucigen	49011-1	Cloning Kit
Expresso Rhamnose Cloning and Expression System Kit, SUMO	Lucigen	49013-1	Cloning Kit
Expresso Rhamnose Cloning and Expression System Kit, C-His	Lucigen	49012-1	Cloning Kit
Glycerol Solution	Sigma-Aldrich	15524-1L-R	Preparation of glycerol stock
L-Rhamnose monohydrate	Sigma-Aldrich	83650	Induction of protein expression
2X Laemmli Sample Buffer	ClearBand	TGS10	SDS-Page analysis
SureCast 40% (w/v) Acrylamide	Invitrogen	HC2040	SDS-Page analysis
SureCast APS	Invitrogen	HC2005	SDS-Page analysis
SureCast TEMED	Invitrogen	HC2006	SDS-Page analysis
10X Running Buffer	ClearBand	TGS10	SDS-Page analysis
Triset al.	BioShop	TRS001.1	SDS-Page analysis and cell lysis
10% SDS	ClearBand	S100	SDS-Page analysis
PageRuler Plus Prestained Protein Ladder	ThermoFisher	26619	SDS-Page analysis
Imidazole	Sigma-Aldrich	56750	Cell lysis
NaCl	Sigma-Aldrich	31434-5Kg-R	Cell lysis
Sodium Phosphate Monobasic Anhydrous	amresco	0571-1Kg	Sodium phosphate buffer for cell lysis
Sodium Phosphate Dibasic Dihydrateet al.	Sigma-Aldrich	04272-1Kg	Sodium phosphate buffer for cell lysis
10-kDa-cut-off centrifugal filter	Amicon®- MERCK	UFC9010	Purification of enzymes