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A Novel Self‐Processing Module Derived from the FrpC Protein of Neisseria meningitidis for a Single‐Step Purification of Free Recombinant Proteins
Author(s) -
Sadilkova Lenka,
Osicka Radim,
Linhartova Irena,
Prochazkova Katerina,
Sebo Peter
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a960-d
Subject(s) - chemistry , maltose binding protein , biochemistry , fusion protein , recombinant dna , flag tag , affinity chromatography , target protein , myc tag , enzyme , gene
Purification of recombinant proteins to homogeneity is often a challenging process and typically requires several chromatographic steps that must be individually optimized for each protein of interest. To overcome this difficulty, a system that enables purification of free recombinant proteins in a single affinity chromatographic step has been developed. The system is based on a 250 amino acid residues long self‐processing module of the FrpC protein of Neisseria meningitidis that is genetically fused at its C‐terminus to an affinity tag enabling simple onestep purification and at its N‐terminus to a protein of interest. Upon binding of the fusion protein to an affinity matrix and washing out of contaminating proteins, specific cleavage between amino acid residues Asp and Pro of the self‐processing module is induced by calcium ions. This results in release of the free protein of interest, having only one extra amino acid residue (Asp) at its C‐terminus. The self‐processing module ‐ affinity tag fusion partner remains trapped on the affinity matrix. This system has been successfully tested with several proteins of interest (adenylate cyclase, chloramphenicol acetyltransferase, β‐galactosidase, maltose‐binding protein, or glutathione‐S‐transferase) and two different affinity tags (chitin‐binding domain, or poly‐His).