Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization
-
Stanislava Bírová
,
Zdenko Levarski
Abstract
Proteoglycan-like (PG) domain of carbonic anhydrase IX (CA IX) is a small-sized acidic domain (8.3 kDa; pI ∼ 3.5) with a very unusual amino acid composition. More than one third of its sequence consists of acidic amino acids and it contains no aromatic residues. It has been revealed that it holds one of the key roles in oncogenetic mechanisms, in which CA IX participates. However, it has not been structurally characterized yet. With these prospects, we developed an expression system of recombinant PG domain production in the form of a fusion protein using thioredoxine (Trx) as the fusion partner in Escherichia coli. We performed three steps during purification process utilizing affinity chromatography, anion-exchange chromatography and gel filtration; and the highest purity we reached was 93.7% with protein concentration 23.3 mg/L. Hypothetical yield of the recombinant protein would be 0.581 mg/L of culture. The applicability of the fusion protein Trx-PG for further structural studies was confirmed by in silico structure prediction tool and ELISA.
Acknowledgements
This publication is the result of the projects implementation: Preparation of biologically active agents based on recombinant proteins (ITMS 26240220048); Centre of excellence for translation research in molecular medicine (ITMS 26240120030) and Comenius University Science Park – 2nd phase (ITMS 26240220086) supported by the Research & Innovation Operational Programme funded by the European Regional Development Fund.
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- Abbreviations
- ANEX
anion-exchange chromatography
- CA
carbonic anhydrase
- CV
column volumes
- EK
enterokinase
- GF
gel filtration
- IPTG
isopropyl β-D-1-thiogalactopyranoside
- LB
Luria-Bertani
- PDB
Protein Data Bank
- PG
proteoglycan-like domain
- TCP
total cell proteins
- Trx
thioredoxine
© 2017 Institute of Molecular Biology, Slovak Academy of Sciences
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