A new procedure for the cloning, expression and purification of the β-carbonic anhydrase from the pathogenic yeast Malassezia globosa, an anti-dandruff drug target

Figures & data

Figure 1. Nucleotide and translated amino acid (bold letters) sequences of MgCA. The open reading frame is indicated by capital letters. Zinc ligands are indicated in red, while the catalytic dyad involved in the activation of the zinc-coordinated water in blue. .

Figure 2. Blast output reporting the first 33 sequences. By going down the list, it is possible to see less than perfect matches, slowly degrading as the corresponding score decreases and the E value increases. The E value is an assessment of the statistical significance of the score. E value close to 1 is a warning that the alignment is not reliable.

Figure 3. SDS-PAGE of the recombinant MgCA purified from E. coli codon plus cells. Lane A, molecular markers, M.W. starting from the top: 250, 150, 100, 75, 50, 37, 25 and 20; Lane B, purified SspCA from His-tag affinity column.

Table 1. Kinetic parameters for the CO₂ hydration reaction catalyzed by MG-CA (GST-fusion protein), MgCA (His-fusion protein), hCA I and II (α-CA from H. sapiens, isoform I and II, respectively) and Can2 (β-CA from C. neoformans).

Cryptonym	Activity level	Class	Kcat (s^−1)	kcat/km M^−1 × s^−1	KI (Acetazolamide) μM
hCA I	Moderate	α	2.0 × 10^5	5.0 × 10^7	0.25
hCA II	Very high	α	1.4 × 10^6	1.5 × 10^8	0.012
Can2	Moderate	β	3.9 × 10^5	4.3 × 10^7	0.010
MgCA	Moderate	β	9.2 × 10^5	8.3 × 10^7	74
MG-CA	Moderate	β	8.6 × 10^5	6.9 × 10^7	76

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4- thiadiazole-2-sulfonamide) are also provided.

Figure 4. MgCA protonogram. The yellow bands correspond to the hydratase activity on the gel responsible for the drop of pH from 8.2 to the transition point of the dye. Incubation time was of 60 s. β-CA is present in the monomeric (29 kDa) form (see text for details). Lane A, molecular markers, M.W. starting from the top: 250, 150, 100, 75, 50, 37, 25 and 20 kDa; Lane B: MgCA; Lane C: commercial bovine CA (bCA).