title: Isolation of trans-acting genes that enhance soluble expression of scFv antibodies in the E. coli cytoplasm by lambda phage display.
abstract: Functional antibody fragments with native disulfide bonds can be expressed in Escherichia coli trxB gor mutant strains having an oxidizing cytoplasm that allows the formation of disulfide bonds. However, expression yields in the cytoplasm are generally lower than those obtained by secretion into the periplasm. We developed a novel methodology for the screening of genomic DNA fragments that enhance expression yields of scFvs in the cytoplasm of trxB gor cells by capitalizing on bacteriophage lambda display. The anti-digoxin 26.10 scFv was displayed on lambda as a fusion to the coat protein gpD. A genomic E. coli library was cloned into lambdagt11 downstream from the lac promoter and used to lysogenize cells transformed with a plasmid encoding the scFv-gpD fusion. Following induction of expression of the cloned gene fragments, phage was prepared and screened for improved functional display via panning against immobilized hapten. Phage exhibiting improved display was isolated after two rounds. One of the isolated clones, encoding the N-terminal domain of the alpha-subunit of RNA polymerase (alpha-NTD), was shown to increase the yield of scFv expressed in soluble form in the cytoplasm.
authors.affiliation: Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712-1064, USA.
authors.forename: Raphael
authors.initials: R
authors.lastname: Levy
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Molineux
authors.forename: Brent L
authors.initials: BL
authors.lastname: Iverson
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015698:Genomic Library; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C050199:digoxin antibodies Fab fragments; D013879:Thioredoxins