Abstract
Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO2 to produce HCO3 − and proton. CAV is primarily involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis by providing bicarbonate ion. Here, we report a new strategy for cloning, expression and purification for CAVA in the bacterial system followed by its biophysical characterization. The cDNA of CAVA, a 801 nucleotide long that encodes a 267-amino acid polypeptide of molecular mass of 30-kDa (excluding signal peptide), was sub-cloned in the expression vector pET21c and transformed into Escherichia coli strain BL21 (DE3) for expression. The recombinant protein was purified in two steps by Ni–NTA and DEAE weak anion-exchange chromatography under native condition from the supernatant, while inclusion bodies (IBs) were used to get protein under the denatured condition with a relatively high yield. CAVA was purified under denatured conditions in a single step using Ni–NTA chromatography. SDS-PAGE showed a band of 30-kDa, which was further confirmed as CAVA by Western blot and MALDI-TOF/MS. We further performed enzyme activity to ensure that both forms of purified proteins are enzymatically active. Measurements of secondary structure of the native, denatured and renatured proteins were carried out using circular dichroism. The purified protein can be further used for structural and biochemical studies.
Highlights
Carbonic anhydrase V (CAV) is a mitochondrial enzyme (Dodgson et al 1980) that catalyzes the hydration of carbon dioxide to produce proton and bicarbonate
We report an alternative method of Carbonic anhydrase VA (CAVA) purification as a major portion of CAVA which was expressed as insoluble protein in the form of inclusion bodies (IBs)
The confirmed plasmid constructed pET21cCAVA was transformed into E. coli BL21 (DE3)
Summary
Carbonic anhydrase V (CAV) is a mitochondrial enzyme (Dodgson et al 1980) that catalyzes the hydration of carbon dioxide to produce proton and bicarbonate. CAV exists in two closely related mitochondrial isoform known as carbonic anhydrase VA (CAVA) and CAVB which have a distinct tissue distribution (Imtaiyaz Hassan et al 2013). CAVA is actively involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis and helps in glucose (Dodgson and Cherian 1989; Parkkila et al 1998), fatty acid and urea synthesis (van Karnebeek et al 2014). CAV is the only mammalian CA which is compartmentalized in a cell organelle and it has tyrosine at 64th position instead of histidine, a catalytic proton shuttle residue which transfers proton from zinc-bound water to buffer (BoriackSjodin et al 1995; Vitale et al 2007). The proton shuttle transport in CAV is still not clear
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