Team:TAS Taipei/Engineering

ENGINEERING SUCCESS

Successful Expression of Part

BBa_K3717016 was added to the iGEM parts registry. We derived the sequence of α-N-Acetylgalactosaminidase (NAGA) from Elizabethkingia meningoseptica (UniProt, 2021) then codon optimized the sequence for E. coli protein expression and attached a 6x Histidine tag (His-tag) downstream of the protein sequence through a glycine-serine linker (GS linker). This formed a new α-N-Acetylgalactosaminidase basic part with a C-Terminal His-tag downstream of the protein sequence (BBa_K3717016).

Figure 1 - Open reading frame for α-N-Acetylgalactosaminidase with C-Terminal 6x Histidine tag (BBa_K3717016)

NAGA is an enzyme that catalyzes the cleavage of the N-acetylgalactosamine off of A type blood antigens such that the remaining sugar can be classified as an H antigen which the anti-A and anti-B antibodies are unable to recognize and hence does not elicit an immune response in the human body (Rahfeld and Withers, 2019). Thus, NAGA can convert A blood types to universal O type.

In order to test our sequence, we attached a T7 promoter + RBS (BBa_K525998) upstream of the open reading frame (ORF) and double terminator (BBa_B0015) downstream of the ORF, which formed our composite part (BBa_K3717013).

Figure 2 - Design of α-N-Acetylgalactosaminidase with T7 Promoter, strong RBS, C-Terminal 6x Histidine tag and Double Terminator Construct (BBa_K3717013)

We tested protein expression of this composite part by transforming the plasmid into BL21 (DE3) E. Coli cells. We grew cultures at 37°C overnight, diluted those cultures, and then grew to OD600 0.5~0.6 at 37°C. We then induced expression with 0.5 mM IPTG and allowed cultures to grow overnight at room temperature. We harvested cells by centrifugation and lysed cell pellets through either sonication or with xTractor Lysis Buffer (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.) supplemented with 20 mM imidazole. We purified our histidine-tagged proteins using Ni sepharose affinity chromatography. We then utilized SDS-PAGE to confirm the size of purified NAGA.

Figure 3 - SDS-PAGE of purified proteins with the T7 promoter α-N-Acetylgalactosaminidase expressing construct (BBa_K3717013). Band matches expected size of 51.7 kDa, proving successful expression and purification.

Our results in Fig 3 indicate a protein band in the purified fraction at roughly 51.7 kDa, which is the molecular weight of the NAGA enzyme with the 6x His tag and GS linker attached. This strongly suggests that NAGA was successfully expressed and purified. We buffer exchanged the purified NAGA enzyme into 0.1 M Sodium Phosphate buffer (pH 7.4) by dialysis due to unfavorable reaction conditions in the eluent, and concentrated the solution to 0.5 mL using centrifugal ultrafiltration.

Successful Function of Part

Colorimetric Substrate Tests

We confirmed the functionality of purified NAGA by using two assays: colorimetric tests and mass spectroscopy.

We obtained a colorimetric substrate for NAGA from Sigma Aldrich (Fig 4). This substrate contains a 4-nitrophenol leaving group, which turns yellow upon successful cleavage in solution. The concentration of 4-nitrophenol was quantified at absorbance 405 nm using a 96-well plate-based assay (Held, BioTek).

Figure 4 - Colorimetric substrates for NAGA.

We performed small-scale colorimetric tests to verify the function of our purified enzymes. To each well, we added 50 μL of 10 mM substrate, 10 μL of enzyme, 30 μL of water, and 10 μL of 10x Glycobuffer 1 (50 mM CaCl2, 500 mM sodium acetate, pH 5.5), a buffer recommended by New England Biolabs to ensure optimal enzyme activity (NEB). Following 2 hours of reaction at room temperature, we diluted the well contents in 1.9 mL of water and took absorbance readings at 405 nm. Our results indicated that NAGA successfully cleaved the substrates (Fig 5). Moreover, we demonstrated the specificity of the enzyme as it did not cleave the substrate for other enzymes.

Figure 5 - Absorbance of reaction solutions in small-scale colorimetric tests verify the functionality and specificity of the enzymes. a) 96-well plates following enzyme reaction. b) absorbance readings of diluted well contents (accounted for dilution). c) buffers and solutions used in substrate and control wells.

To quantify the activity of NAGA, we performed enzyme reactions at various substrate concentrations and measured the absorbance at different time intervals over a constant time period (Fig 6). We used the averages from at least three independent trials to calculate the Michaelis–Menten constant for enzyme efficiency (see enzyme kinetics model in modeling). The results of our colorimetric substrate tests show that NAGA enzyme is specific and functions as expected.

Figure 6 - Absorbance over time for various NAGA substrate concentrations

Mass Spectroscopy Trisaccharide Antigen Tests

Given that the colorimetric results were positive, we wanted to further test the enzyme’s specificity by using trisaccharides as substrates. These trisaccharides contain three monosaccharides that have identical chemical structures to the A and B antigens found on RBCs. To test a proof of concept functionality of NAGA in cleaving the A-antigen trisaccharide, we carried out a reaction of the enzyme and its trisaccharide dissolved in 1X GlycoBuffer (New England BioLabs), deionized water, and purified BSA for 1 hour at 37°C. The A-antigen trisaccharides were kindly supplied by Professor Dr. Todd Lowary from the Institute of Biological Chemistry at Academic Sinica (Lowary 2010).

For the enzyme tests, a non-specific trisaccharide served as a negative control and A-antigen trisaccharide specific to NAGA served as the experimental unit (Table 1).

Table 1 - Experimental setup for enzyme-antigen tests.

After the reaction, the reaction solution was passed through C18 columns to minimize impurities from the reaction solution. We then evaluated the flow through solution with a mass spectrometer to measure peaks in molar mass in order to determine the compounds present in the reaction solution. The original molar mass of the A-antigen is 713.35 g/mol; therefore, after cleavage, we expected two fragments with molar masses of 510.27 g/mol and 203.08 g/mol to form. (Fig 7).

Figure 7 - Molecular structure and mass of A-antigen trisaccharides and subsequent products following enzymatic cleavage.

The results shown by the mass spectrometer confirm the functionality of NAGA. The experimental unit shows the presence of the cleaved fragment from the reaction with A-antigen trisaccharide, indicated by the peak at 533 g/mol in the mass spectrum (Fig 8). The negative control confirms the specificity of NAGA, since it did not cleave the B-antigen trisaccharide, as indicated by the peak at 695 g/mol (Fig 9). Throughout all readings of the mass spectrum, the molar mass of the peaks have been increased by 23 g/mol due to a sodium adduct. Unrelated peaks/background noise can be attributed to compounds present in the buffer solution.

Figure 8 - α-N-Acetylgalactosaminidase cleaves A-antigen trisaccharide.Mass spectrum of α-N-Acetylgalactosaminidase and A-antigen trisaccharide reaction solution (experimental unit, flow through). The peak at 533g/mol shows the presence of the cleaved fragment from the reaction of the α-N-Acetylgalactosaminidase enzyme and A-antigen trisaccharide.

Figure 9 - Negative Control: Mass spectrum of α-N-Acetylgalactosaminidase and B-antigen trisaccharide reaction solution (negative control, flow through). The negative control confirms the specificity of α-N-Acetylgalactosaminidase, as the B-antigen trisaccharide was not cleaved, indicated by the peak at 695g/mol

Mass spectroscopy results from the reaction between NAGA and A-antigen trisaccharide substrate demonstrate its ability to cleave its specific trisaccharides, demonstrating the proof of concept that the enzyme is able to cleave the A blood group antigens.

References

Rahfeld, Peter, and Stephen G. Withers. “Toward Universal Donor Blood: Enzymatic Conversion of A and B to O Type.” Journal of Biological Chemistry, vol. 295, no. 2, Jan. 2020, pp. 325–34. DOI.org (Crossref), https://doi.org/10.1074/jbc.REV119.008164.

XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10. Held, Paul. “Kinetic Analysis of β-Galactosidase Activity Using the PowerWave™ HT and Gen5™ Data Analysis Software .” BioTek, 16 Feb. 2007.

Biolabs, New England. “Typical Reaction Conditions for α-N-Acetylgalactosaminidase (P0734).” New England Biolabs: Reagents for the Life Sciences Industry, https://international.neb.com/protocols/2013/01/10/typical-reaction-conditions-p0734.

Meloncelli, Peter J., and Todd L. Lowary. “Synthesis of Abo Histo-Blood Group Type I and II Antigens.” Carbohydrate Research, vol. 345, no. 16, 16 Sept. 2010, pp. 2305–2322., https://doi.org/10.1016/j.carres.2010.08.012.

UniProtKB - A4Q8F7 (GH109_ELIME). UniProt, 2 June 2021, www.uniprot.org/uniprot/A4Q8F7. Accessed 20 Oct. 2021.