Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA

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• 作者：Danish Idrees ; Sudhir Kumar ; Syed Abdul Arif Rehman ; Samudrala Gourinath…
• 关键词：Carbonic anhydrase VA ; Protein expression ; Gluconeogenesis ; Enzyme assay ; Circular dichroism ; Protein purification
• 刊名：3 Biotech
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：6
• 期：1
• 全文大小：2,395 KB
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• 作者单位：Danish Idrees (1)
  Sudhir Kumar (2)
  Syed Abdul Arif Rehman (2)
  Samudrala Gourinath (2)
  Asimul Islam (1)
  Faizan Ahmad (1)
  Md. Imtaiyaz Hassan (1)
  
  1. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India
  2. School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110025, India
  
• 刊物主题：Biotechnology; Agriculture; Cancer Research; Bioinformatics; Stem Cells; Biomaterials;
• 出版者：Springer Berlin Heidelberg
• ISSN：2190-5738

文摘

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. Keywords Carbonic anhydrase VA Protein expression Gluconeogenesis Enzyme assay Circular dichroism Protein purification