Difference between revisions of "Part:BBa K3717007"

Revision as of 09:54, 18 October 2021

α-N-Acetylgalactosaminidase with N-Terminal 6x Histidine tag

α-N-Acetylgalactosaminidase catalyzes the cleavage of the terminal N-acetylgalactosamine of A type blood antigens such that the resultant antigen can be classified as an 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. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.

Figure 1: α-N-Acetylgalactosaminidase with 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-N-Acetylgalactosaminidase 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. The composite part 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.

Cells were then harvested after the overnight induction and lysed them either through sonication or with xTractor Lysis Buffer (XTractorTM Buffer & XTractor Buffer Kit User Manual, n.d.) spiked with 500mM Imidazole stock (to a final concentration of 20mM in the lysate solution). The Histidine tagged proteins were then purified using Ni sepharose affinity chromatography. SDS-PAGE was then utilized to confirm sizes of purified proteins.

Our results indicate no protein bands, showing that our protein purification was unsuccessful.

Sequence and Features