We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cisprolines. We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA.FkpA is one of three soluble peptidylprolyl cis,trans-isomerases (PPIase) 1 in the periplasm of Escherichia coli, in addition to PpiA (RotA) and SurA (1). It was first described as being similar to the macrophage infectivity potentiator of Legionella species (2). It is homologous to the FK506-binding proteins and was subsequently shown to belong to the E regulon and thus to be inducible under stress conditions reviewed in Ref. 6). Although the fkpA deletion mutant has been shown to be viable (1, 2), FkpA has been proposed to have a general folding-assisting function in the periplasm (Refs. 1 and 5; reviewed in Ref. 6). This idea was based on an fkpA deletion causing up-regulation of E and thus stimulation of degP transcription and, in addition, FkpA's own induction by extracytoplasmic stress (5) and its ability to restore near-normal E -dependent response when overexpressed (1).In the accompanying paper (7), FkpA was identified in a selection system as a factor improving the functional expression of antibody scFv fragments in the bacterial periplasm. This was achieved by overexpressing a library of E. coli proteins in a phagemid, on which a poorly expressing antibody was encoded for phage display, and panning for functional antibody. In vivo overexpression experiments provided further evidence that the yield of functional antibody scFv fragments produced in the periplasm is increased upon coexpression of FkpA. Interestingly, the effect was also observed with antibody fragments devoid of cis-prolines (7). In antibody scFv fragments, the variable domain of the heavy chain (V H ) is connected via a linker to the variable domain of the light chain (V L ). Antibody V H domains have n...

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