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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- Zoology
- Identifying white spots on the roadmap of Late Pleistocene and Holocene palaeolimnology in Slovakia: Review and future directions
- Cellular and Molecular Biology
- Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization
- Botany
- Functional and morphological traits of epiphytic lichens in the Western Carpathian oak forests reflect the influence of air quality and forest history
- Botany
- Clonostachys rosea associated with ponderosa and Coulter pine needles in Slovakia
- Botany
- First signs of old-growth structure and composition of an oak forest after four decades of abandonment
- Botany
- Factors responsible for the distribution of invasive plant species in the surroundings of railway areas. A case study from SE Poland
- Botany
- The role of lipids and polysaccharides in model root mucilage with implications for the surface activity of the rhizosphere
- Botany
- Physiological and proteomic changes in Zizania latifolia under chilling stress
- Botany
- Novel polymorphic EST-based microsatellite markers characterized in lettuce (Lactuca sativa)
- Cellular and Molecular Biology
- Identification of a HSP40 gene involved in planarian regeneration
- Zoology
- A new species of Bothropolys and a new record of Lithobius magnitergiferous (Lithobiidae) from the Qinghai-Tibet Plateau, China
- Zoology
- Myriapod (Chilopoda, Diplopoda) communities in hedgerows of upland agricultural landscape
- Zoology
- A new cockroach, with bipectinate antennae,(Blattaria: Olidae fam. nov.) further highlights the differences between the Burmite and other faunas
- Zoology
- Pre-winter larval activity and feeding behavior of Erebia aethiops and E. cassioides in Austrian Alps
- Zoology
- Genetic structure of Apis mellifera carnica in Slovakia based on microsatellite DNA polymorphism
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- Celluar and Molecular Biology
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- The growth inhibitory effects of garlic polysaccharide combined with cis-dichlorodiamine platinum on human HepG2 cells
- Erratum
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