Systematic screening of soluble expression ofantibody fragments in the cytoplasm of E. coli

Microbial Cell Factories

Research

Chunguang Wang¹  Sohvi Hörkkö²  Mirva J. Saaranen³  Lloyd W. Ruddock³  Johanna Veijola³  Anna Gaciarz³  Yuko Uchida³ 

[1] Department of Medical Microbiology and Immunology and Medical Research Center, University of Oulu, Oulu, Finland;Department of Medical Microbiology and Immunology and Medical Research Center, University of Oulu, Oulu, Finland;Nordlab Oulu, Oulu University Hospital, Oulu, Finland;Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland;

关键词: (3-10): Antibody fragments;    Disulfide bonds;    Fab;    scFv;    Cytoplasm;    Escherichia coli;   

DOI  :  10.1186/s12934-016-0419-5

 received in 2015-11-03, accepted in 2016-01-11,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundDisulfide bonds are the most common structural, post-translationalmodification found in proteins. Antibodies contain up to 25 disulfide bondsdepending on type, with scFv fragments containing two disulfides and Fabfragments containing five or six disulfide bonds. The production of antibodyfragments that contain native disulfide bonds can be challenging, especially ona large scale. The protein needs to be targeted to prokaryotic periplasm or theeukaryotic endoplasmic reticulum. These compartments are specialised fordisulfide bond formation, but both compartments have limitations.ResultsHere we show that the introduction into the cytoplasm of a catalystof disulfide bond formation and a catalyst of disulfide bond isomerizationallows the efficient formation of natively folded scFv and Fab antibodyfragments in the cytoplasm of Escherichiacoli with intact reducing pathways. Eleven scFv and eleven Fabfragments were screened and ten of each were obtained in yields of >5 mg/Lfrom deep-well plates. Production of eight of the scFv and all ten of the Fabshowed a strong dependence on the addition of the folding factors. Yields ofpurified scFv of up to 240 mg/L and yields of purified Fab fragments of up to42 mg/L were obtained. Purified fragments showed circular dichroism spectraconsistent with being natively folded and were biologically active.ConclusionsOur results show that the efficient production of soluble,biologically active scFv and Fab antibody fragments in the cytoplasm ofE. coli is not only possible, but facile.The required components can be easily transferred between different E. coli strains.

【 授权许可】

CC BY   
© Gaciarz et al. 2016