Cloning and functional characterization of a γ-carbonic anhydrase (CA) gene from the gametophytes of Saccharina japonica

In this study, the full-length cDNA and DNA of a γ-type carbonic anhydrase (CA) gene was obtained from the gametophyte of Saccharina japonica by the rapid amplification of cDNA ends (RACE) technique. The results showed that the cDNA of this gene was 1 618 bp in length, encoding a protein consisting of 305 amino acids, and the genomic DNA was 11 812 bp long, with 6 introns, so that the open reading frame (ORF) was divided into 7 exons. It had a gamma-CA domain and a unique LβH domain. The Neighbor-joining and the Maximum Likelihood phylogenetic tree constructed from the deduced amino acid sequences of 36 CAs showed that the cloned CA was clustered with other γ-CAs. Therefore, the gene was designated Sjγ-CA. In order to understand the function of the encoded protein, the ORF of Sjγ-CA was subcloned and ligated into the expression vector pET-28a to generate pET28a-SiγCA. Subsequently, this construct was introduced into Escherichia coli BL21 for the heterologous expression of target protein. The recombinant Sjγ-CA was purified by affinity chromatography. After SDS-PAGE electrophoresis, Western blotting analysis and mass spectrometry, the purified recombinant Sjγ-CA was identified. The activity of rSjγ-CA in the hydration reaction of CO₂ and \rmHCO_3^ - was detected by electrode method and its specific activity was 0.82 U/mg. The hydrolysis of p-nitrophenyl acetate was detected by spectrophotometer, but no esterase activity was detected. Sjγ-CA was thus identified functionally. This study provides a basis for the subcellular localization of Sjγ-CA in gametophyte and sporophyte cells or tissues of S. japonica.