title: Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli.
abstract: BACKGROUND
  Disulfide bonds are the most common structural, post-translational modification found in proteins. Antibodies contain up to 25 disulfide bonds depending on type, with scFv fragments containing two disulfides and Fab fragments containing five or six disulfide bonds. The production of antibody fragments that contain native disulfide bonds can be challenging, especially on a large scale. The protein needs to be targeted to prokaryotic periplasm or the eukaryotic endoplasmic reticulum. These compartments are specialised for disulfide bond formation, but both compartments have limitations.
  
  
  RESULTS
  Here we show that the introduction into the cytoplasm of a catalyst of disulfide bond formation and a catalyst of disulfide bond isomerization allows the efficient formation of natively folded scFv and Fab antibody fragments in the cytoplasm of Escherichia coli with intact reducing pathways. Eleven scFv and eleven Fab fragments were screened and ten of each were obtained in yields of >5 mg/L from deep-well plates. Production of eight of the scFv and all ten of the Fab showed a strong dependence on the addition of the folding factors. Yields of purified scFv of up to 240 mg/L and yields of purified Fab fragments of up to 42 mg/L were obtained. Purified fragments showed circular dichroism spectra consistent with being natively folded and were biologically active.
  
  
  CONCLUSIONS
  Our results show that the efficient production of soluble, biologically active scFv and Fab antibody fragments in the cytoplasm of E. coli is not only possible, but facile. The required components can be easily transferred between different E. coli strains.
authors.affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland. annagaciarz@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Gaciarz
authors.affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland. johanna.veijola@oulu.fi.
authors.forename: Johanna
authors.initials: J
authors.lastname: Veijola
authors.affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland. yuko.uchida@oulu.fi.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland. mirva.saaranen@oulu.fi.
authors.forename: Mirva J
authors.initials: MJ
authors.lastname: Saaranen
authors.affiliation: Department of Medical Microbiology and Immunology and Medical Research Center, University of Oulu, Oulu, Finland. chunguang.wang@oulu.fi.
authors.forename: Chunguang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Medical Microbiology and Immunology and Medical Research Center, University of Oulu, Oulu, Finland. sohvi.horkko@oulu.fi.|Nordlab Oulu, Oulu University Hospital, Oulu, Finland. sohvi.horkko@oulu.fi.
authors.forename: Sohvi
authors.initials: S
authors.lastname: Hörkkö
authors.affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland. Lloyd.Ruddock@oulu.fi.
authors.forename: Lloyd W
authors.initials: LW
authors.lastname: Ruddock
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D057127:Single-Chain Antibodies / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D057127:Single-Chain Antibodies
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