Difference between revisions of "Part:BBa K3717015"

Latest revision as of 08:34, 21 October 2021

α-Galactosidase with C-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 C-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) downstream of the α-Galactosidase sequence preceded by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717015) 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_K3717012) was assembled through DNA synthesis by IDT.

However, cells transformed with the plasmids had problems growing on culture plates and therefore, we were unable to commence protein purification.

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.

Sequence and Features