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Efficient purification of His-tagged human epididymis protein 4 (HE4) using magnetic TiO2 nanotube carrier

Autoři: Kupčík Rudolf | Macák Jan | Řehulka Pavel | Bílková Zuzana

Rok: 2015

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	Efficient purification of His-tagged human epididymis protein 4 (HE4) using magnetic TiO2 nanotube carrier	Synthesis of recombinant proteins presents essential importance in life sciences. High purity of recombinant proteins is one of the most important prepositions for use as therapeutic or diagnostic agents. Human epididymis protein 4 (HE4) was designated as a serum biomarker for ovarian cancer. Recombinant HE4 protein can be utilized e.g. for immunization of rabbit and also for subsequent purification of polyclonal antibodies. These polyclonal anti-HE4 IgG molecules as well as recombinant HE4 protein are then exploited for development of electrochemical immunosensor for HE4 tumor marker detection. Definitely, purity requirements in the case of antigen for immunization or other applications are extremely high. Immobilized-Metal Affinity Chromatography (IMAC) is commonly used for separation of recombinant proteins but protein is usually obtained with insufficient purity. In our work, the innorganic composite was used for isolation His-tagged recombinant HE4 from the model mixture of proteins. Particular isolation steps were analysed using Tris-Tricine gel electrophoresis with sbsequent silver staining. Analysis demonstrates high selectivity of the composite for His-tagged proteins and in combination with tailored binding and elution conditions (imidazole in combination with Na2HPO4) offer efficient separation of recombinant polyhistidine-tagged protein. SEM morphological characterizations of composite were also performed. Compare to IMAC, imidazole itself seems to be ineffective to elute His-tagged proteins from carrier which can be advantageously used for removing weakly bound proteins including histidines in their natural sequence. The achieved isolation specificity is able to have direct positive impact on the increased purification efficiency of protein with very promising implications in life science, research and diagnostics.

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