Expression, Single-Step Purification and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific beta-Glucosidase

Varování

Publikace nespadá pod Fakultu sportovních studií, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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ZOUHAR Jan NANAK Elizabeth BRZOBOHATÝ Břetislav

Rok publikování 1999
Druh Článek v odborném periodiku
Časopis / Zdroj Protein Expression and Purification
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Obor Genetika a molekulární biologie
Klíčová slova beta-glucosidase; immobilized metal affinity chromatography; perfusion matrix; refolding
Popis Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)6Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)6Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after refolding process was confirmed by Km determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.
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