Part:BBa_K3717008
Endo-β-Galactosidase with N-Terminal 6x Histidine tag
Endo-β-galactosidase catalyzes the cleavage of A and B type blood antigen trisaccharides such that the remaining sugar can be classified as a precursor to the 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, the enzyme converts both A and B blood types to universal O type.
Figure 1: Endo-β-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 Endo-β-galactosidase sequence followed by a glycine-serine linker (GS linker) to form our open reading frame (ORF) (BBa_K3717008) 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_K3717011) 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(DE3) 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 no protein bands, showing that our protein purification of BBa_K3717011 was unsuccessful.
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
- 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 2035
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 660
Illegal SapI.rc site found at 2848
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