Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody F(ab) fragment without the need for an affinity tag.
Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant F(ab) fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli. Adsorption of the F(ab) fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a His(6) tag or Strep tag or of the human or murine nature of the C(H)1 and C(L) domains (subclass IgG1/kappa). Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F(ab) fragment at high purity. Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F(ab) fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer. Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F(ab) fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix. This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F(ab) fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system.
Fiedler
M
M
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, D-85350, Germany.
Skerra
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D007075:Immunoglobulin M / Q000737:chemistry*; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D010649:Phenylalanine