Production of stabilized antibody fragments in the E. coli bacterial cytoplasm and in transiently transfected mammalian cells

Racheli Birnboim-Perach, Yehudit Grinberg, Lilach Vaks, Limor Nahary, Itai Benhar

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv. The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Number of pages26
StatePublished - 2019

Publication series

NameMethods in Molecular Biology


  • Fab
  • Maltose-binding protein (MBP)
  • Monoclonal antibody (mAb)
  • Single chain variable fragment (scFv)
  • Tobacco Etch Virus (TEV) protease

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics


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