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Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA
Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO(2) to produce HCO(3) (−) and proton. CAV is primarily involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis by providing bicarbonate ion. Here, we report a n...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705057/ https://www.ncbi.nlm.nih.gov/pubmed/28330086 http://dx.doi.org/10.1007/s13205-015-0334-1 |
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author | Idrees, Danish Kumar, Sudhir Rehman, Syed Abdul Arif Gourinath, Samudrala Islam, Asimul Ahmad, Faizan Imtaiyaz Hassan, Md. |
author_facet | Idrees, Danish Kumar, Sudhir Rehman, Syed Abdul Arif Gourinath, Samudrala Islam, Asimul Ahmad, Faizan Imtaiyaz Hassan, Md. |
author_sort | Idrees, Danish |
collection | PubMed |
description | Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO(2) to produce HCO(3) (−) 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. |
format | Online Article Text |
id | pubmed-4705057 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-47050572016-01-11 Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA Idrees, Danish Kumar, Sudhir Rehman, Syed Abdul Arif Gourinath, Samudrala Islam, Asimul Ahmad, Faizan Imtaiyaz Hassan, Md. 3 Biotech Original Article Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO(2) to produce HCO(3) (−) 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. Springer Berlin Heidelberg 2016-01-07 2016-06 /pmc/articles/PMC4705057/ /pubmed/28330086 http://dx.doi.org/10.1007/s13205-015-0334-1 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Original Article Idrees, Danish Kumar, Sudhir Rehman, Syed Abdul Arif Gourinath, Samudrala Islam, Asimul Ahmad, Faizan Imtaiyaz Hassan, Md. Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title | Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title_full | Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title_fullStr | Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title_full_unstemmed | Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title_short | Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA |
title_sort | cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase va |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705057/ https://www.ncbi.nlm.nih.gov/pubmed/28330086 http://dx.doi.org/10.1007/s13205-015-0334-1 |
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