Ameloblastin (Ambn) is an intrinsically disordered protein (IDP) with a specific function of forming heterogenous homooligomers. The oligomeric function is led through a specific sequence encoded by exon 5 of Ambn. Due to the IDP character of Ambn to form oligomers, protein purification is subject to many challenges. Human ameloblastin (AMBN) and its two isoforms, I and II have already been purified as a recombinant protein in a bacterial expression system and functionally characterized in vitro. However, here we present a new purification protocol for the production of native AMBN in its original formation as a homooligomeric heterogeneous IDP. The purification process consists of three chromatographic steps utilizing His-tag and Twin Strep-tag affinity chromatography, along with size exclusion and reverse affinity chromatography. The presented workflow offers the production of AMBN in sufficient yield for in vitro protein characterizations and can be used to produce both AMBN isoforms I and II.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Namesti 2 16000 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Namesti 2 16000 Prague Czech Republic; 2nd Faculty of Medicine Charles University 150 06 Prague 5 5 Uvalu 84 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo Namesti 2 16000 Prague Czech Republic; Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

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Proteolytic profiles of two isoforms of human AMBN expressed in E. coli by MMP-20 and KLK-4 proteases