Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization
| Autor: | Stanislav Stuchlík, Silvia Pastorekova, Stanislava Birova, Ján Turňa, Zdenko Levarski, Ivana Vidlickova |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
In silico Size-exclusion chromatography Plant Science medicine.disease_cause Biochemistry law.invention 03 medical and health sciences Affinity chromatography law Genetics medicine Molecular Biology Escherichia coli Ecology Evolution Behavior and Systematics biology Chemistry Cell Biology Fusion protein 030104 developmental biology Proteoglycan Yield (chemistry) Recombinant DNA biology.protein Animal Science and Zoology |
| Zdroj: | Biologia. 72:1240-1246 |
| ISSN: | 1336-9563 0006-3088 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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