Part:BBa_K3717016
α-N-Acetylgalactosaminidase with C-Terminal 6x Histidine tag
α-N-Acetylgalactosaminidase catalyzes the cleavage of the terminal N-acetylgalactosamine of A type blood antigens such that the resultant antigen 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 [1]. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.
Figure 1. α-N-Acetylgalactosaminidase with C-Terminal 6x His-Tag and GS linker.
Construct Design
We derived the sequence of α-N-Acetylgalactosaminidase from Elizabethkingia meningoseptica [2] and optimized the sequence for E. coli protein expression. We then attached a 6x histidine tag (6x His-Tag) downstream of the α-N-Acetylgalactosaminidase sequence preceded by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717016) for purification purposes. We flanked our open reading frame with a T7 promoter + RBS (BBa_K525998) upstream of the open reading frame and a double terminator (BBa_B0015) downstream of the sequence. This composite part (BBa_K3717013) was assembled through DNA synthesis by IDT.
Characterization
Protein Expression and Purification
We transformed synthesized plasmids 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 [3] supplemented with 20 mM imidazole. We purified our histidine-tagged proteins using Ni sepharose affinity chromatography. We then utilized SDS-PAGE to confirm the sizes of purified proteins.
Our results indicate a protein band at roughly 51.7 kDa, which is the molecular weight of our α-N-Acetylgalactosaminidase enzyme with the 6x His tag and GS linker attached, proving that our α-N-Acetylgalactosaminidase (Part: BBa_K3717013) was expressed and purified.
Figure 2. SDS-PAGE of purified proteins with T7 promoter α-N-Acetylgalactosaminidase expressing construct (BBa_K3717013). Red triangles indicate expected size for the part.
References
1. 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.
2. UniProtKB - A4Q8F7 (GH109_ELIME). UniProt, 2 June 2021, www.uniprot.org/uniprot/A4Q8F7. Accessed 20 Oct. 2021.
3. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 529
- 1000COMPATIBLE WITH RFC[1000]
| None |