Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

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 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 species^8,9. 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 conditions^9,10. This novel glycoside hydrolase enzyme can cleave the N-N′-diacetylchitobiose parts found in the N-glycans^10,11. The activity of EndoBI-1 is not affected by core fucosylation and different reaction conditions such as high temperature, pH, reaction time, etc^3,11,12. This unique characteristic of Bifidobacterial glycoside hydrolases provides a promising tool for producing N-glycans from glycoprotein-rich substrates such as bovine colostrum^13,14.

Several chemically and enzymatically developed deglycosylation methods have been widely used to obtain N-glycans and O-glycans from glycoproteins^2,15. 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 difficult^19,20. 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 methods^16,22,23. 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 charge^24,25,26,27. 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-glycans^10,11,28. 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 glycoproteins^30,31. 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).

Representative 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 (Supplementary Table 1). Forward and reverse primers for N-His Kan Vector were designed as; F: 5'-CAT CAT CAC CAC CAT CAC XXX₂ XXX₃ XXX₄ XXX₅ XXX₆ XXX₇ XXX₈ (XXX₂-XXX₈ represents codons 2 through 8 of the target coding region) and R: 5'-GTG GCG GCC GCT CTA TTA XXX_n XXX_n-1 XXX_n-2 XXX_n-3 XXX_n-4 XXX_n-5 XXX_n-6 (XXX_n - XXX_n-6 represents the sequence complementary to the last 7 codons of the target coding region), respectively. Forward and reverse primers for C-His Kan Vector were designed as F: 5'-GAA GGA GAT ATA CAT ATG XXX₂ XXX₃ XXX₄ XXX₅ XXX₆ XXX₇ XXX₈ (XXX₂-XXX₈ represents codons 2 through 8 of the target coding region) and R: 5'-GTG ATG GTG GTG ATG ATG XXX_n XXX_n-1 XXX_n-2 XXX_n-3 XXX_n-4 XXX_n-5 XXX_n-6 (XXX_n - XXX_n-6 represents the sequence complementary to the last 7 codons of the target coding region), respectively. Forward and reverse primers for N-His SUMO Kan Vector were designed as; F: 5'- CGC GAA CAG ATT GGA GGT XXX₂ XXX₃ XXX₄ XXX₅ XXX₆ XXX₇ XXX₈ (XXX₂-XXX₈ represents codons 2 through 8 of the target coding region) and R: 5'-GTG GCG GCC GCT CTA TTA XXX_n XXX_n-1 XXX_n-2 XXX_n-3 XXX_n-4 XXX_n-5 XXX_n-6 XXX_n - XXX_n-6 (XXX_n - XXX_n-6 represents the sequence complementary to the last 7 codons of the target coding region), respectively. Targeted genes were amplified by PCR. Each reaction mixture was a total of 50 µL containing 25 µL of master mix, 2 µL of a template (Bifidobacterial cells), 1 µL of Forward Primer, 1 µL of Reverse Primer, 21 µL of DNase/RNase-free water. PCR reaction was initiated with 95 °C for 5 min to release genomic DNA by cell lysis. Then, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min as well as a final extension step at 72 °C for 10 min. PCR products were visualized by DNA gel electrophoresis to control the concentration and the purity of the inserts (Figure 1). The gel images showed that each amplification was successfully completed as there was only one clear band for each insert. Each PCR amplicon was measured by a fluorometer to determine the concentration of the PCR product (Table 2).

Bacterial Strains	Origin	Accession Number	Locus Tag	Number of Base Pair
Bifidobacterium pullorum subsp. pullorum	Chicken feces	WP052122780.1	OU11_RS07620	1226
Bifidobacterium pullorum subsp. saeculare	Rabbit feces	KFI88990.1	BSAE_0444	2811
Bifidobacterium kashiwanohense	Child feces	BAQ30016.1	BBKW_1881	1302
Bifidobacterium pseudocatenulatum	Infant feces	BAR04361.1	BBPC_1683	1314
Bifidobacterium bohemicum	Bumblebee digestive tract	KFI46963.1	BBOH_0438	1650

Table 1: Information about target genes and their origin.

Figure 1: PCR amplification of targeted genes by three different vector primer sets. (A) N-His (B) C-His (C) N-His SUMO. Lanes 1-5 represent OU11_RS07620, BSAE_0444, BBKW_1881, BBPC_1683, and BBOH_0438 amplification, respectively. Please click here to view a larger version of this figure.

Samples	N-His	N-His SUMO	C-His	Final Volume
B. pullorum subsp. pullorum (OU11_RS07620)	9	14.2	10.76	50 µL
B. pullorum subsp. saeculare (BSAE_0444)	24	12.66	11.74	50 µL
B. kashiwanohense (BBKW_1881)	14.6	35.9	20.6	50 µL
B. pseudocatenulatum (BBPC_1683)	20	35.3	21.9	50 µL
B. bohemicum (BBOH_0438)	30.8	32.5	10.4	50 µL

Table 2: Measurement of DNA concentrations by a fluorometer (ng/µL).

The transformation of His-tagged PCR products and vectors (N-His Kan, C-His Kan, and N-His SUMO Kan) into chemically competent E. coli cells were performed by the heat shock method. Firstly, 1-3 µL (25 to 100 ng) of the PCR products and 2 µL of vectors were added into 40 µL of chemically competent E. coli. The mixtures were stirred gently with a pipette tip, transferred into 15 mL centrifuge tubes, and incubated on ice for 30 min. Heat shock was performed in a 42 °C water bath for 45 s for the transformation of the PCR products and the vectors into the cells. The heat-shocked cells were then returned quickly to ice for 2 min. 960 µL of recovery medium, which allows the cells to repair, was added to each tube and incubated at 37 °C for 1 h at 250 rpm. 100 µL of the cells were plated on LB + agar plates containing 30 µg/mL of kanamycin. In this experiment, chemically competent E. colicells were only used as a negative control. All plates were incubated overnight at 37 °C. After the transformations, 20-60 colonies were obtained as expected. The growth of these cells on a medium containing LB + agar + 30 µg/mL of kanamycin was indicated that the kanamycin-resistant vectors were successfully transferred to the cells. Successful transformants were confirmed by the colony PCR method. Firstly, one part of each colony was transferred into a PCR tube for PCR amplification, and the other part was inoculated into 5 mL of LB + kanamycin medium in a 15 mL falcon tube to be used in case of positive results. PCR amplification was performed using 25 µL of master mix, 1 µL of forward primer, 1 µL of reverse primer (sequencing primers provided by the manufacturer), 23 µL of dH₂O at 95 °C 5 min for the release of genomic DNA by lysing cells, a total of 40 cycles of 95 °C 30 s for initial denaturation, 60 °C 30 s for annealing and 72 °C 1 min for elongation, and 72 °C 10 min for the final extension. 5 µL of PCR products mixed with a loading dye at a ratio of 1:1, were run on a 1% agarose gel at 100 V for 70 min and visualized with a gel documentation system. Consequently, positive transformants for all target genes were separated from the unsuccessful colonies. When the gel image was examined, it was observed that almost all colonies were successful in transformation of the target genes (Figure 2). Glycerol stocks were prepared from the liquid cultures of the successful colonies.

Figure 2: Gel image obtained after Colony PCR. Lanes 1-5, 6-10, 11-15, and 16-18 represent OU11_RS07620, BBKW_1881, BBPC_1683, and BBOH_0438 amplification, respectively. Please click here to view a larger version of this figure.

Protein production was initially carried out on a small scale (8 mL), then it was produced in large quantities using 250 mL of medium. Firstly, protein production was initiated with 1% inoculation of LB medium containing 30 µg/mL of kanamycin, allowing the chemically competent E. coli cells optical density to reach approximately 0.5-0.6. Protein expression was induced by the addition of L-rhamnose with a final concentration of 0.2%. After overnight incubation, the bacteria pellets were collected by centrifugation. The cell lysis method was used to break the cell membranes to release DNA, RNA, or protein from the induced cells. The homogenization of the cells was performed by adding lysis buffer and protease inhibitor. The sonication method was also used for secondary homogenization. Then, the cell debris was removed from the lysed cells by centrifugation.

Protein purification was performed using the batch method. Firstly, an equilibration buffer was added into both the Ni-NTA resin and the proteins to ensure that the 6xHis-tagged proteins interact effectively with the resin. Ni-NTA resin was added to 6xHis-tagged proteins and incubated to attach the proteins to the resin. 6xHis tagged proteins bound to nickel resins, while other natural proteins flowed through the system. The mixture containing the proteins attached to the resin was washed with the Wash Buffer for the removal of potential pollution by repeated centrifugation processes. The proteins were separated from the resin with Elution Buffer containing high imidazole. The purified protein was concentrated using a 15 mL, 10 kDa cut-off centrifugal filter. The list of purified enzyme codes is listed in Table 3. Consequently, the targeted proteins were successfully purified with a high yield by the batch purification method (Figure 3).

Enzymes	N-His	N-His SUMO	C-His
OU11_RS07620	N1	S1	C1
BSAE_0444	N2	S2	C2
BBKW_1881	N3	S3	C3
BBPC_1683	N4	S4	C4
BBOH_0438	N5	S5	C5

Table 3: Codes of N-His tagged, N-His SUMO tagged and C-His tagged purified enzymes.

Figure 3: SDS-PAGE analysis of purified enzymes. N-His tagged, C-His tagged, and N-His-SUMO tagged enzymes were successfully purified with high yield by the batch purification method. Please click here to view a larger version of this figure.

Supplementary Table 1: List of primers used for gene amplification and sequencing. Primer sets for the target genes were designed based on the manufacturer's guidelines. Please click here to download this Table.

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 requirement of restriction enzymes. In this system, cloning can be performed by mixing the amplified gene with the cell and the vector (provided with the kit) without the need for enzymatic ligation. That's why it is called in vivo cloning; recombination between the ends of the PCR product and the vector occurs in the cell. The linear vector in this system contains 18 nucleotide-long sticky ends at both ends and these sequences are not complementary to each other. Owing to this feature, the vector completely prevents the risk of self-ligation or inverted insertion of sequences. Primers required for the amplification of the target gene are designed according to these ends for the strong binding. The N-His, C-His, and N-His SUMO vectors provided in the cloning kit facilitate instant cloning of target genes with amino or carboxyl-terminal 6xHis affinity tags that provide fast and easy affinity purification of proteins under native or denaturing conditions.

Although the transformation success efficiency of this cloning system is above 95%, one of the few disadvantages is the competent cells, which cannot be produced in typical laboratory conditions. The linearized vector containing competent cells is also only provided by the manufacturer. Thus, the storage and their application should be performed carefully. We have observed that heat shock transformation should be performed in 15 mL falcon tubes compared to 0.2 mL PCR tubes, which are commonly preferred by conventional molecular cloning methods.

After molecular cloning, protein production is increased by using L-rhamnose and its purification is performed by binding to nickel columns with 6xHistidine tag in three different vectors. To improve the purification efficiency, an N-His SUMO tag has been tested in this study. SUMO protein is known to improve the solubility of proteins and this enhances the recovery of purified protein³². However, we have not observed significant increases in recovery when comparing the N-His SUMO tag and N- and C-Histidine tags.

Protein purification is a critical step for obtaining enzymes with high purity. Impurities recovered with the purified protein might mislead downstream experiments as well as efficiency calculations or activity assays in the case of enzymes. Successful purification is often considered to be 95% or more based on visual observation of the DNA gel electrophoresis result. Although a similar process can be applied for structurally similar proteins, each step of the purification process should be optimized for higher protein production efficiency and purity. One of the important steps in this process is the application of washing buffer to remove unwanted proteins and other compounds. The concentration of the washing buffer can contain 10 to 50 mM imidazole. While a low concentration of imidazole can cause impurities in enzyme solution, a high concentration of imidazole might diminish the recovery of proteins from the column. Therefore, at least three different concentrations of imidazole should be tested for the highest efficiency of protein production with the highest purity (optimized based on SDS-PAGE gel profiling).

With this fast and efficient molecular cloning-protein purification technique, we would enable to clone the enzymes from different sources and use them in various applications in several studies related to microbiology, food applications, and microbiome. One possible future direction with these techniques is the integration of microbiome-based enzymes into in vitro digestion models or using them in some products such as infant formulas and/or milk.

Materials

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