Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli

Supaluk Krittanai, Waraporn Putalun, Seiichi Sakamoto, Hiroyuki Tanaka, Thaweesak Juengwatanatrakul, Gorawit Yusakul

Research output: Contribution to journalArticlepeer-review

Abstract

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, VH–CH1 could associate VL–CL into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of VH–CH1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of VL–CL, the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

Original languageEnglish
Pages (from-to)4519-4529
Number of pages11
JournalMolecular biology reports
Volume47
Issue number6
DOIs
Publication statusPublished - Jun 1 2020

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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