'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems

Protein Eng Des Sel. 2016 Apr;29(4):149-57. doi: 10.1093/protein/gzw001. Epub 2016 Feb 21.


A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.

Keywords: Escherichia coli; cell-free protein synthesis; expression system; fragment of antigen binding; leucine zipper.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell-Free System
  • Escherichia coli / genetics
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / genetics*
  • Immunoglobulin Fab Fragments / metabolism
  • Leucine Zippers / genetics*
  • Protein Engineering / methods*
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / metabolism


  • Immunoglobulin Fab Fragments
  • Recombinant Proteins