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A Novel Carrier Based on TiO2 Suitable for Isolation of His-tagged Recombinant Proteins and Peptides
Autoři: Kupčík Rudolf | Macák Jan | Krulišová Pavla | Řehulka Pavel | Bílková Zuzana
Rok: 2015
Druh publikace: ostatní - přednáška nebo poster
Strana od-do: nestránkováno
Tituly:
Jazyk Název Abstrakt Klíčová slova
eng A Novel Carrier Based on TiO2 Suitable for Isolation of His-tagged Recombinant Proteins and Peptides To achieve a high level of purity in the purification of recombinant proteins for therapeutic or analytical application, usually several chromatographic steps are used. Many of the recombinant proteins are synthesized with suitable affinity tags (e.g. His-tag) to use the advantage of the selectivity of the affinity chromatography. In the case of a complex mixture of proteins or a clarified cell extract, there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity. Introducing of materials with higher isolation efficiency can significantly reduce the number of accompanied purification steps, increase the yield and decrease the overall costs.After optimizing of experimental conditions for purification of His-tagged compounds on peptide level using MALDI-Orbitrap MS measurements, separation of protein mixtures containing His-tagged protein prior to and after purification procedure was performed. SDS-PAGE analysis of fractions eluted from the presented nanomaterial contained recombinant protein present in significantly higher purity compared to eluent obtained from application of the widely used commercial IMAC (Ni-NTA) particles. The obtained results indicate a high selectivity of the used nanomaterial. A stronger interaction between this carrier and corresponding His-tagged protein was also observed. This allows to use higher concentrations of eluting agents which may reduce the non-specific binding of other compounds and thus the higher purity of the isolated protein may be obtained. The fast magnetic response, robustness and simple handling are other important advantages of new TiO2 based magnetic nanomaterial. nanomaterial, titanium dioxide, mass spectrometry, His-tag

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