The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines.
The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Plückthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Plückthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity.
Bothmann
H
H
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Pluckthun
A
A
D001435:Bacteriophages / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D020104:Immunophilins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase; D019897:Periplasm / Q000201:enzymology*; D011392:Proline / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins; D011994:Recombinant Proteins; D011392:Proline; D020104:Immunophilins; D019696:Peptidylprolyl Isomerase; C068254:FkpA protein, E coli