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 purificatio n of a recombinant Feb fragment of the antibody IN-1 from the periplasmic p rotein fraction of Escherichia coil. 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 Hiss 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 fract ions with the correctly assembled, heterodimeric F-ab fragment at high puri ty. Interestingly, the single substitution of an alanine residue with pheny lalanine in the CDR-L1 of the F-ab fragment significantly enhanced the rete ntion on the column so that quantitative elution necessitated prolonged app lication of a low-salt buffer. Our findings suggest that TAC is generally s uitable for the isolation of bacterially produced F-ab fragments and suppor t the notion that aromatic side chains play an important role in the intera ction with the affinity matrix. This method should prove valuable in the pr oduction 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 t he central nervous system, (C) 1999 Academie Press.