Part:BBa_K3717006
α-Galactosidase with N-Terminal 6x Histidine tag
α-Galactosidase catalyzes the cleavage of the galactose off of B type blood antigens such that the remaining sugar can be classified as a 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-Galactosidase converts B blood types to universal O type.
Figure 1. α-Galactosidase with N-Terminal 6x His-Tag and GS linker.
Construct Design
We derived the sequence of α-Galactosidase from Bacteroides fragilis [2] and optimized the sequence for E. coli protein expression. We then attached a 6x Histidine Tag (6x His-Tag) upstream of the α-Galactosidase sequence followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717006) 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_K3717009) 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 took samples pre-induction and post-induction and examined them by SDS-PAGE.
Our results indicate a protein band at roughly 69.7 kDa, which is the molecular weight of our α-Galactosidase enzyme with the 6x His tag and GS linker attached, proving that our α-Galactosidase (Part: BBa_K3717009) was expressed and purified.
Figure 2. SDS-PAGE of purified proteins with the T7 promoter α-Galactosidase expressing construct (BBa_K3717009). 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 208
Illegal AgeI site found at 511 - 1000COMPATIBLE WITH RFC[1000]
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