Cloning and Characterization of putative alpha family Carbonic Anhydrase from Leishmania major

Singh, Shalini (2014) Cloning and Characterization of putative alpha family Carbonic Anhydrase from Leishmania major. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Carbonic anhydrases (EC 4.2.1.1) (CA) are metalloenzymes which catalyze the reversible conversion of carbon dioxide and water to form bicarbonate ions and protons. The major function of the CAs is to maintain acid-base homeostasis in the living organisms. However, they also play an important part in other physiological and pathological processes like gluconeogenesis, lipogenesis, ureagenesis, tumorigenicity and growth, survival of various pathogens. Analysis of Leishmania major genome suggests that it encodes for two distinct putative CA-like proteins which belong to the β and α classes of CAs respectively. Sequence alignment of the α-CA (LmCA2) with human α-CAs shows that the metal binding site is conserved, while the other catalytic residues are partially conserved. Expression of LmCA2 mRNA transcripts was confirmed by reverse transcriptase PCR (RT-PCR). This gave us the impetus to PCR amplify the LmCA2 gene from the Leishmania genome, clone it in a pET28a cloning/expression plasmid vector and over-express it in commercial strains of Escherichia coli. However, the presence of hydrophobic N-terminal signal sequence and transmembrane domain near the C-terminal hindered the expression of this eukaryotic protein in a bacterial system. Hence, we constructed LmCA2_ΔN/pET28a and LmCA2_ΔC/pET28a clones by deleting these hydrophobic patches. Only the LmCA2_ΔN protein was expressed in E.coli BL21(DE3) cells upon 0.5mM IPTG induction and this ~70kDa protein was verified by Coomassie staining using protein markers of known molecular weight. Most of the protein is present in the insoluble fraction of the cell. To study the function of LmCA2 protein in L.major cells, we checked for its subcellular localization. We cloned LmCA2 in pXG-GFP vectors and expressed the GFP-tagged LmCA2 protein in wild type Leishmania cells. Preliminary localization studies suggest that LmCA2 is targeted to the plasma membrane or any cell organelle membrane. Thus, here we report the presence of first α-CA in L.major cells which along with LmCA1 (β-CA) could play an important role in acid acclimation of the parasite.

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