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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 glycosylation1,2. 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 proteins through N-acetylglucosamine (GlcNAc) in a particular amino acid sequence AsN-X-Ser/Thr and a less common one, AsN-X-Cys (cysteine) (where X might be any amino acid except proline)3,4. The basic N-glycan core consists of two HexNAc and three mannose residues. Further elongation of this common core with other monosaccharides via glycosyltransferase and glycosidase enzymes determines the type of N-glycans based on the degree of branching and the type of linkage5. N-glycans are generally grouped into three main classes: high mannose (HM), complex type (CT), and hybrid (HY)6.

N-glycans are indigestible compounds by the host organisms due to the lack of glycoside hydrolase enzymes. These compounds reach the small/large intestine in an undigested form where thousands of different bacterial species utilize them, and they can act as prebiotics by promoting specialized gut microbes, especially Bifidobacterium species7. Recent findings showed that N-glycans selectively stimulate the growth of certain bacterial species8,9. N-glycans released from bovine milk glycoproteins selectively stimulated the growth of Bifidobacterium longum subspecies infantis (B. infantis), which is a crucial Bifidobacterial species in the infant's gut, but other bifidobacterial species such as Bifidobacterium animalis (B. animalis) did not utilize these compounds9. In addition, a recent in vivo study demonstrated that 19 unique N-glycans from milk lactoferrin and immunoglobulins selectively stimulate the growth of B. infantis8. Especially, B. infantis possess a genomic capability for glycan cleavage and metabolism. An Endo-β-N-acetylglucosaminidase (EndoBI-1), which belongs to glycosyl hydrolase family 18, recombinantly produced from B. infantis ATCC 15697 showed a high activity on milk glycoproteins in in vitro conditions9,10. This novel glycoside hydrolase enzyme can cleave the N-N′-diacetylchitobiose parts found in the N-glycans10,11. The activity of EndoBI-1 is not affected by core fucosylation and different reaction conditions such as high temperature, pH, reaction time, etc3,11,12. This unique characteristic of Bifidobacterial glycoside hydrolases provides a promising tool for producing N-glycans from glycoprotein-rich substrates such as bovine colostrum13,14.

Several chemically and enzymatically developed deglycosylation methods have been widely used to obtain N-glycans and O-glycans from glycoproteins2,15. Chemical methods are widely used in glycobiology for deglycosylation of glycoproteins because of their ease of use, low cost, and high substrate specificity16. The most common chemical deglycosylation methods are β-elimination and hydrazination17. Among these methods, β-elimination is based on the principle of cleavage of glycans from glycoproteins by exposure of glycoproteins to alkaline conditions. The released glycans can be degraded during the process due to the β-elimination reactions, but this problem can be prevented using reducing agents such as sodium borohydride (NaBH4)18,19,20. There are different limitations in the β-elimination method. The reductive agents convert glycans to alditols, prevent them from binding a fluorophore or chromophore. Thus, challenging to monitor glycan release becomes difficult19,20. Because of the high salt content in the cleaning step of the method, elution might result in sample losses20. Another method for releasing glycan from glycoproteins is the hydrazine method based on the principle of the hydrolysis reaction following the addition of anhydrous hydrazine to the glycoprotein. Since it allows for controlling the isolation of glycans by changing reaction conditions such as temperature, the hydrazination method has been widely used in glycobiology21. Chemical deglycosylation can also be carried out using the anhydrous formulation of hydrogen fluoride and trifluoroacetic acid, in addition to other chemical deglycosylation methods16,22,23. The enzymatic release of N-glycans from glycoproteins is commonly performed by peptidyl-N-glycosidases (PNGases) that generally release N-glycans, regardless of their size and charge24,25,26,27. Similar to the chemical deglycosylation methods, the enzymatic deglycosylation process has different challenges. PNGases show activity in the presence of several detergents used, which increase the enzyme accessibility to the glycans. However, these harsh treatments might disrupt the native glycans and the remaining polypeptide structures28. PNGases may not cleave the glycans when there is a fucose linked to N-acetylglucosamine29. Various endoglycosidases such as F1, F2, and F3 show more activity on the native proteins than PNGases. These endoglycosidases have low activity on the multiple-antennary glycans, whereas heat-resistant novel EndoBI-1 is effective in all types of N-glycans10,11,28. Regarding the limitations of the current methods, it is obvious that novel enzymes are still required for an effective glycan release without any restrictions. For this purpose, Bifidobacterial species, which have a large genomic island encoding various glycoside hydrolases enzymes, enable cleaving N-glycans from glycoproteins30,31. Within the scope of this context, the overall aim of this study is to discover new glycosidases from the various Bifidobacterial species. To recombinantly produce these enzymes, different fusion tags are intended to enhance their production as well as their activity.

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 the mixture by pipetting up and down.
      NOTE: The master mix (Lucigen) used for PCR contains Taq DNA Polymerase with high purity and high activity and can work at higher temperatures for reliable amplification of templates up to 5 kb.
    3. Set the PCR program as follows: initial denaturation step at 95 °C for 5 min for the release of genomic DNA, then 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 1 min, and the final extension at 72 °C for 10 min.
    4. Check the PCR products by agarose gel electrophoresis method with a gel documentation systemafter being run at 100 V for 60 min on the 1% agarose gel. Mix the PCR products and DNA ladder with the loading dye by mixing at a ratio of 1:5 (5 µL of PCR product + 1 µL of loading dye and 5 µL of 1 kb DNA ladder + 1 µL of loading dye) to load on the gel (Figure 1).
    5. Measure the DNA concentrations of PCR products using a fluorometer before the molecular cloning step.
      NOTE: Concentrations of PCR products should be in the range required for molecular cloning (25-100 ng/µL).
  2. Preparation of Lysogeny Broth (LB) agar medium for molecular cloning
    1. Dissolve 12.5 g of LB and 6 g of agarose in 500 mL of dH2O and autoclave the LB agar medium (sterilization at 121 °C for 20 min).
    2. Dissolve 15 mg of kanamycin with 1 mL of dH2O and store at -20 °C.
    3. After autoclaving, add 1 mL of kanamycin into the bottle containing the sterile 500 mL of LB agar medium. The final concentration of kanamycin is 30 µg/mL. Pour 25 mL of LB agar kanamycin media into each plate in the laboratory cabinet.
  3. Heat shock transformation of chemically competent E. coli cells
    1. Add 1-3 µL (25 to 100 ng) of the PCR products for each strain into the tube, including 40 µL of chemically competent E. coli cells. Then, add 2 µL of the vector DNA to the same tube. Stir gently with the pipette tip and transfer the mixtures to 15 mL centrifuge tubes.
      NOTE: Perform this step on ice. Do not pipette up and down to mix to avoid air bubbles and inadvertently warming cells.
    2. Incubate the tubes containing the competent cells and DNA on ice for 30 min. Apply heat shock to the mixture in a 42 °C water bath for 45 s. Put these tubes on ice immediately and incubate for 2 min.
    3. Add 960 µL of the Recovery Medium, used for the rapid recovery of cells after molecular cloning, to the cells in the tubes and incubate the tubes at 250 rpm for 1 h at 37 °C in a shaking incubator.
    4. Plate 100 µL of transformed cells on LB agar plates containing 30 µg/mL of kanamycin. Use only chemically competent E. coli cells as a negative control.
      NOTE: Put LB agar plates containing 30 µg/mL of kanamycin prepared in step 1.2.2 to the incubator at 37 °C before using.
    5. Incubate all plates overnight at 37 °C under ambient atmosphere (Figure 2).
  4. Preparation of LB medium for colony PCR
    1. Dissolve 7.5 g of LB with 300 mL of the dH2O in a bottle and autoclave the LB medium (sterilization at 121 °C for 20 min).
    2. Dissolve 9 mg of kanamycin (30 µg/mL) with 1 mL of dH2O and put at -20 °C. After autoclaving, add 1 mL of kanamycin into the bottle containing the sterile 300 mL of LB medium. Store the liquid culture medium at +4 °C until using it.
  5. Screening of transformants by colony PCR
    1. To confirm all transformants carry the recombinant genes, select colonies randomly and amplify the target genes by PCR using the sequencing primers supplied with the cloning kit.
    2. Perform all the steps on ice and pre-chill all PCR tubes and 15 mL tubes before use.
    3. Using a pipette tip, transfer half of a selected colony to the PCR tube for each sample. Take another half of the colony with the pipette tip and put it into the 15 mL tube containing 5 mL of LB+kanamycin liquid medium (prepared in step 1.4). Vortex the 15 mL tubes and incubate the liquid cultures at 250 rpm at 37 °C overnight in a shaking incubator.
    4. Put 50 µL of PCR reaction mixture (25 µL of master mix, 1 µL of forward primer, 1 µL of reverse primer, 23 µL of DNase/RNase-free distilled water) into all PCR tubes and disperse the cells by pipetting up and down gently.
    5. Set the PCR program at 95 °C for 5 min for the release of genomic DNA by lysing bacterial cells, a total of 40 cycles of 95 °C for 30 s for initial denaturation, 60 °C for 30 s for annealing, 72 °C for 1 min for elongation, and 72 °C for 10 min for the final extension.
    6. Check the PCR products by gel electrophoresis method after being run at 100 V for 60 min on the 1% agarose gel (Figure 3). The details of DNA gel electrophoresis are described in step 1.1.3.
    7. Prepare 15% glycerol stocks of the successful transformants. Put 500 µL of 60% glycerol stock in the cryotubes and add 1,500 µL of the liquid culture of the successful transformants. Store prepared stocks at -80 °C.

2. L-rhamnose induction of protein expression

  1. Prepare a preculture with 1 L of LB liquid medium containing 30 µg/mL of kanamycin.
  2. Put 8 mL of the LB medium in 50 mL centrifuge tubes. Use one of the tubes as a negative control containing only the liquid medium. For 20% L-rhamnose as stock, dissolve 0.5 g of L-rhamnose with 2.5 mL of dH2O and store at -20 °C until using it.
  3. Put 10 µL of the bacterial stocks into the centrifuge tubes containing 8 mL of liquid media. Vortex them gently and incubate at 37 °C for overnight in the shaking incubator.
  4. Pour 250 mL of LB liquid medium into a sterilized 2 L Erlenmeyer flask.
  5. Inoculate 2.5 mL of the overnight liquid culture into a 2 L flask containing LB liquid medium at a ratio of 1:100 between the flask and the medium, and incubate at 37 °C and150 rpm for 4 h in the shaking incubator.
  6. Measure the optical density at 600 nm (OD600) for the bacterial cells by a spectrophotometer. When the cells reach the optical density of 0.5-0.6, add 2.5 mL of 20% rhamnose (final concentration is 0.2%) to the 250 mL of LB culture and incubate at 37 °C overnight at 250 rpm in the shaking incubator.
  7. Transfer the liquid culture into the 5 x 50 mL tubes, centrifuge samples 3724 x g for 15 min at +4 °C and discard the supernatant. Store the pellets at -20 °C until the purification step.
    ​NOTE: To evaluate protein expression with SDS-PAGE, collect 1 mL of uninduced (when cultures at an optical density 600 nm of 0.5-0.6, without L-rhamnose) culture as control and 1 mL of induced culture (after overnight incubation) as induced sample. Microcentrifuge all samples at 12,000 x g for 1 min and resuspend uninduced and induced samples with 50 µL and 100 µL of SDS-PAGE loading buffer, respectively.

3. Cell lysis of chemically competent E. coli cells containing His-tagged enzymes

  1. Prepare lysis buffer pH 8.0 (50 mM Tris-HCl, 200 mM NaCl, 1 mM imidazole, 1% SDS), equilibration buffer pH 7.4 (20 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), wash buffer pH 7.4 (20 mM NaH2PO4, 300 mM NaCl, 25 mM imidazole), and elution buffer pH 7.4 (20 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole).
  2. Place the 50 mL tubes containing the cell pellets at -80 °C for 15 min to freeze. Then, remove the pellets from -80 °C and thaw at room temperature.
  3. Add 5 mL of dH2O to the pellets and dissolve by pipetting up and down. Centrifuge at 3724 x g for 15 min at +4 °C and discard the supernatant.
  4. For 50 mL culture pellets, add 6,300 µL of lysis buffer and 63 µL of EDTA-free halt protease inhibitor cocktail (1:100 ratio) into the pellets and dissolve by pipetting up and down. Incubate them on ice for 30 min, vortex every 10 min.
  5. Set the pulse mode of the sonicator as 10 s ON and 59 s OFF, and the amplitude as 37%. Place the tube in a beaker containing ice and immerse the probe of the sonicator in the tube.
    NOTE: The probe should be immersed completely without touching any side of the tube.
  6. After the sonication process (6 pulses for 10 s with 1 min cooling), centrifuge the samples at 3,724 x g for 45 min at +4 °C. Next, collect all the supernatant parts in a tube and centrifuge at 3,724 x g for 5 min at +4 °C.
  7. Collect the supernatant into a tube and take 100 µL of the sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in step 4.7. Measure the protein concentrations of the samples using a fluorometer.

4. Purification of His-tagged enzymes by batch method

  1. Add 1 mL of Ni-NTA resin to a centrifuge tube and centrifuge for 2 min at 700 x g. Carefully remove the tube and discard the supernatant formed.
  2. Add 2 mL (twice the resin volume) of the equilibration buffer into the tube and mix well until the resin is fully suspended. Centrifuge the tube for 2 min at 700 x g and carefully remove and discard the buffer.
  3. Mix the protein extract and equilibration buffer in a ratio of 1:1 in a centrifuge tube. Add the mixture to the tube containing resin and put it on a shaker at 150 rpm for 30 min. Centrifuge the tube for 2 min at 700 x g and discard the supernatant.
  4. Wash the resin with 5 mL of wash buffer and centrifuge the tube for 2 min at 700 x g. Repeat the washing step until the concentration of the supernatant decreases to the baseline.
  5. Add 1 mL of elution buffer into the tube to elute bound His-tagged proteins. Centrifuge the tube for 2 min at 700 x g. Save the supernatant and take 100 µL for SDS-PAGE analysis in step 4.7. Repeat the elution step three times. Measure the concentrations of each supernatant by using a fluorometer.
  6. Collect all the supernatants in the 10 kDa cut-off tube and centrifuge it for 2 min at 700 x g. Repeat centrifugation until the volume of the supernatant decreases to 200 µL by pipetting up and down occasionally. Store the purified proteins at -20 °C and take 100 µL for SDS-PAGE analysis in step 4.7.
    NOTE: Protein concentration should be measured and, if the concentration is low, centrifuge until it increases.
  7. SDS-PAGE analysis of the purified proteins
    1. Prepare a 4% stacking gel (40% acrylamide/bisacrylamide, 1 M Tris pH 6.8, 10% SDS, 10% ammonium persulfate, TEMED, dH2O) and 12% resolving gel (40% acrylamide/bisacrylamide, 1 M Tris pH 8.8, 10% SDS, 10% ammonium persulfate, TEMED, dH2O).
    2. Mix the sample with 2x Laemmli sample buffer in a ratio of 1:1 and incubate at 95 °C for 5 min to denature the proteins.
      NOTE: Protein concentration of samples should be measured before loading, and the loaded volume will be based on their concentration for equal loading.
    3. Add 1x running buffer into the tank and load the samples and the protein ladder into the wells. Run the proteins firstly at 80 V, and raise the current to 120 V when the proteins move from the stacking gel to the resolving gel.
    4. Put the gel in coomassie blue staining dye and put it in a shaker for 30 min. Wash the gel with a destaining solution (250 mL dH2O + 50 mL acetic acid (HOAc) + 200 mL methanol), and take the image (Figure 4).

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...

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study is supported by TUBITAK #118z146 and Uluova Süt Ticaret A.Ş (Uluova Milk Trading Co.).

Materials

NameCompanyCatalog NumberComments
EconoTaq PLUS 2X Master MixLucigen30035-1Amplification of target genes (PCR)
DNase/RNase-free distilled waterInvitrogen10977035Amplification of target genes (PCR)
Safe-Red Loading DyeabmG108-RDNA gel electrophoresis
1 kb Plus DNA LadderGoldBioD011-500DNA gel electrophoresis
Qubit protein assay kitInvitrogenQ33211Measurement of DNA concentration
LB Broth, Miller (Luria-Bertani)amrescoJ106-2KGBacterial culture media
AgaroseInvitrogen16500-500Bacterial culture mediaet al.
Kanamycin MonosulfateGoldBioK-120-5Antibiotic in bacterial culture media
Expresso Rhamnose Cloning and Expression System Kit, N-HisLucigen49011-1Cloning Kit
Expresso Rhamnose Cloning and Expression System Kit, SUMOLucigen49013-1Cloning Kit
Expresso Rhamnose Cloning and Expression System Kit, C-HisLucigen49012-1Cloning Kit
Glycerol SolutionSigma-Aldrich15524-1L-RPreparation of glycerol stock
L-Rhamnose monohydrateSigma-Aldrich83650Induction of protein expression
2X Laemmli Sample BufferClearBandTGS10SDS-Page analysis
SureCast 40% (w/v) AcrylamideInvitrogenHC2040SDS-Page analysis
SureCast APSInvitrogenHC2005SDS-Page analysis
SureCast TEMEDInvitrogenHC2006SDS-Page analysis
10X Running BufferClearBandTGS10SDS-Page analysis
Triset al.BioShopTRS001.1SDS-Page analysis and cell lysis
10% SDSClearBandS100SDS-Page analysis
PageRuler Plus Prestained Protein LadderThermoFisher26619SDS-Page analysis
ImidazoleSigma-Aldrich56750Cell lysis
NaClSigma-Aldrich31434-5Kg-RCell lysis
Sodium Phosphate Monobasic Anhydrousamresco0571-1KgSodium phosphate buffer for cell lysis
Sodium Phosphate Dibasic Dihydrateet al.Sigma-Aldrich04272-1KgSodium phosphate buffer for cell lysis
10-kDa-cut-off centrifugal filterAmicon®- MERCKUFC9010Purification of enzymes

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Recombinant ProductionBifidobacterial EndoglycosidasesN glycan ReleaseProtein GlycosylationPrebiotic CompoundsGut MicrobiomeGlycoproteinsMolecular CloningEnzymatic ProtectionProtein Purification

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