Difference between revisions of "Part:BBa K3717007"

Latest revision as of 08:29, 21 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 [1]. Thus, α-N-Acetylgalactosaminidase can convert A blood types to universal O type.

Figure 1. α-N-Acetylgalactosaminidase with N-Terminal 6x His-Tag and GS linker.

Construct Design

We derived the sequence of α-N-Acetylgalactosaminidase from Elizabethkingia meningoseptica [2] and optimized the sequence for E. coli protein expression. We 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_K3717007) 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_K3717010) 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 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 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) [3]. We purified the Histidine tagged proteins using Ni sepharose affinity chromatography. We then utilized SDS-PAGE to confirm the sizes of purified proteins.

Our results indicated no protein bands, showing that our protein purification of BBa_K3717010 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. UniProtKB - A4Q8F7 (GH109_ELIME). UniProt, 2 June 2021, www.uniprot.org/uniprot/A4Q8F7. Accessed 20 Oct. 2021.

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 258
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1278

@@ Line 19: / Line 19: @@
 <b><font size="+0.5"> Protein Expression and Purification </font></b>
-We tested protein expression of the composite parts by transforming our plasmids into BL21(DE3) <i>E. coli</i> 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 took samples pre-induction and post-induction and examined them by SDS-PAGE.
+We tested protein expression of the composite parts by transforming our plasmids into BL21(DE3) <i>E. coli</i> 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 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) [3]. We purified the Histidine tagged proteins using Ni sepharose affinity chromatography. We then utilized SDS-PAGE to confirm the sizes of purified proteins.