Difference between revisions of "Part:BBa K2539201"

Revision as of 03:43, 11 October 2018

ALDH2*2 + 6XHIS Expressing Construct

ALDH2*2 is the mutant form of human mitochondrial aldehyde dehydrogenase (ALDH2), the enzyme responsible for converting acetaldehyde, a toxic intermediate, into acetate in alcohol metabolism. The mutant ALDH2*2 (common in East Asia) is slow to convert acetaldehyde, a toxic intermediate, into acetate (Chen et al., 2014).

For protein purification, we added a HIS-tag (6xHIS) to the N-terminus of the ALDH2*2 sequence (basic part is BBa_K2539250). 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_K2539201 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_K2539201 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, and we saw a band around that size in the eluate (red arrowhead in figure below). This shows that we were able to purify HIS-tagged ALDH2*2.

SDS-PAGE results show protein content at different steps of protein purification. HIS-tagged ALDH2*2 has an expected size of around 56 kDa. A band can be seen in the eluate (red arrowhead) around 56 kDa.

Enzyme Activity

We tested the enzyme activity of HIS-tagged ALDH2*2 (BBa_K2539201). When ALDH2 converts acetaldehyde into acetate, NADH is produced. To test the ability of recombinant ALDH2*2 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 wild type HIS-ALDH2*1 (BBa_K2539101) and the mutant, HIS-ALDH2*2 (BBa_K2539201). Purified HIS-ALDH2*2 did not have any effect on NADH concentration, while purified HIS-ALDH2*1 significantly increased NADH levels.

Purified HIS-ALDH2*2 has no effect on acetaldehyde. 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

Chen CH, Ferreira JCB, Gross ER, Mochly-Rosen D. (2014). Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities. Physiol Rev. 94(1):1-34.

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.

Sequence and Features