Difference between revisions of "Part:BBa K2539101"

Latest revision as of 10:25, 11 October 2018

6xHIS-ALDH2*1 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.

Construct Design

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.

Purified HIS-ALDH2*1 has a much higher activity level compared to purified HIS-ALDH2*2. The enzymatic activity of purified HIS-ALDH2*1 and HIS-ALDH2*2 were tested at 25°C. A negative control containing only elution buffer (from the protein purification process) was also included (gray). HIS-ALDH2*1 steadily metabolized more acetaldehyde compared to both HIS-ALDH2*2 and the negative control, both of which did not seem to have any effect. The error bars represent standard error.

References

Harimech PK, Hartmann R, Rejman R, del Pino P, Rivera-Gila P, Parak WJ. (2015). Encapsulated enzymes with integrated fluorescence-control of enzymatic activity. J. Mater. Chem. B. 3, 2801-2807.

McComb RB, Bond LW, Burnett RW, Keech RC, Bowers, GN Jr. (1976). Determination of the molar absorptivity of NADH. Clin Chem. 22(2): 141–150.