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Revision as of 11:01, 10 October 2018

ALDH2*1 + 6XHIS Expressing Construct

ALDH2*1 is the wild type form of human mitochondrial aldehyde dehydrogenase (ALDH2), the enzyme responsible for converting acetaldehyde, a toxic intermediate, into acetate in alcohol metabolism.

For protein purification, we added a HIS-tag (6xHIS) to the N-terminus of the ALDH2*1 sequence (basic part is BBa_K2539150). This was flanked by a strong promoter and strong RBS combination (BBa_K880005) and a downstream double terminator (BBa_B0015) to maximize expression.

PCR Check Results

The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.

PCR check for BBa_K2539101 using VF2 and VR primers. Using these primers, PCR produced a band at the expected size of 2.1 kb.

Characterization

Protein Purification:

E. coli carrying BBa_K2539101 was lysed and run through a nickel column (GE Healthcare, 11-0033-99). HIS-tagged proteins should bind to the column, which contains nickel ions. SDS-PAGE was used to check protein content at different steps of the purification process: lysed cell sample, flow-through after the wash buffer, and final eluate containing the purified protein (shown below). HIS-tagged ALDH2 should be around 56 kDa. In the initial cell lysate lane, there is a band around 50 kDa. This band disappears in the wash buffer flow-through lane, and reappears In the eluate. This shows that we are able to purify HIS-tagged ALDH2*1.

SDS-PAGE results show the proteins present at different steps of protein purification. A band around 56 kDa in the cell extract (green) and the eluate (red), but not present in the wash buffer flow through lane (yellow), matches our expected HIS-tagged ALDH2*1 (BBa_K2539101).

Enzyme Activity Test:

We tested the enzyme activity of HIS-tagged ALDH2*1 (BBa_K2539101). When ALDH2 converts acetaldehyde into acetate, NADH is produced. To test the ability of recombinant ALDH2*1 to metabolize acetaldehyde, we used reagents from a kit (Megazyme, K-ACHYD) to quantify the amount of NADH produced by taking absorbance readings at 340 nm. This wavelength is highly absorbed by the reduced form, NADH, but not the oxidized form, NAD+ (Harimech et al., 2015; McComb et al., 1976). High absorbance values would indicate more conversion of acetaldehyde into acetate.

We saw a clear difference between the activity levels of HIS-ALDH2*1 (BBa_K2539101) and HIS-ALDH2*2 (BBa_K2539201). Purified HIS-ALDH2*2 did not have any effect on NADH, while purified HIS-ALDH2*1 significantly increased NADH levels.