Difference between revisions of "Part:BBa K3717006"

Latest revision as of 01:41, 10 October 2023

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 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 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.

***Corrections***

iGEM ULaval 2023

We noted that this enzyme was misidentified as GH110b throughout the wiki. It is however the GH110a from Bacteroides fragilis after verification in the original article (Liu et al., 2008) and Blast search. We used and added the GH110b from B. fragilis in the registry; BBa_K4726002. It was shown that both GH110a and GH110a have similar affinity and catalytic activity to the B type antigen. Although, GH110a is more stringent and uses only towards branched B antigen, whereas GH110b was shown to be active towards both branched and linear B antigen (Liu et al., 2008).

Furthermore, the Uniprot entry cited here is not referring to the same protein (sequences are different). The right Uniprot entry is Q5L7M8.

Liu QP, Yuan H, Bennett EP, Levery SB, Nudelman E, Spence J, Pietz G, Saunders K, White T, Olsson ML, Henrissat B, Sulzenbacher G, Clausen H. Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen. J Biol Chem. 2008 Mar 28;283(13):8545-54. doi: 10.1074/jbc.M709020200. Epub 2008 Jan 28. PMID: 18227066; PMCID: PMC2417185.

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 - Q5LIM3 (Q5LIM3_BACFN). UniProt, 2 June 2021, www.uniprot.org/uniprot/Q5LIM3. Accessed 20 Oct. 2021.

3. XTractorTM Buffer & xTractor Buffer Kit User Manual. (n.d.). 10.

Sequence and Features