Difference between revisions of "Part:BBa K3717006"

Revision as of 14:27, 18 October 2021

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 6x N-Terminal His-Tag and GS linker

Construct Design

We optimized the DNA sequence for expression in E. coli, 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 tested protein expression of the composite parts by transforming our plasmids into BL21 E. Coli cells. We grew an overnight culture of the BL21 cells with our plasmids then diluted our cells to a standardized OD600 of ~0.1 and let it grow until an OD600 of 0.5~0.6. The diluted cultures of OD600 0.5~0.6 were then induced for expression with 0.5 M IPTG stock (to a final concentration of 0.5mM in the culture) and allowed to grow and induce overnight at room temperature.

We harvested the cells after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution) [2]. We purified the Histidine tagged proteins using Ni sepharose affinity chromatography [2]. 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.

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

Sequence and Features