• Periplasmic expression of novel tetravalent single chain antibody-streptavidin fusion protein and optimization of expression conditions
  • Aref Farokhi-Fard,1 Fatemeh Davami,2,* Farzaneh Barkhordari,3 Elham Bayat,4
    1. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
    2. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
    3. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran
    4. Biotechnology research center, Pasteur institute of Iran, Tehran, Iran


  • Introduction: Tetravalent single chain antibody-streptavidin fusions ((scFv-SA)4) are promising bispecific agents with potential applications in several therapeutic and diagnostic approaches. Some of these structures have been reached to phase 2 clinical trials for radio-immunotherapy of blood malignancies. Bacterial expression of such constructs can be very challenging because of their inherent structural complexity (several disulfide bonds and tetrameric entity which need to correct assembly of 12 domains and leads to high molecular weight (MW) of the fusion) as well as biotin depletion-associated cellular toxicity of streptavidin moiety. In previous works, usually the full length SA (amino acid residues 1-159) or core SA (13-139) has been utilized for the design. The former structure increases the MW as well as tendency to aggregation while the latter, increases the toxic effect on the host (because of enhanced affinity of SA toward cellular biotin). For the first time we periplasmically expressed a novel fusion using amino acids 13-159 of SA which lacks amino-terminal sequence of the native SA (theoretically lower MW and lower aggregation tropism) but contains carboxy-terminal sequence of the native SA (lower affinity to biotin and therefore lower toxicity). We used scFv sequence of murine anti IL-1RAP antibody as a model. Moreover, a detailed optimization on cell growth/induction condition was performed to improve the expression level of the fusion.
  • Methods: Western blot (WB) analysis (with HRP-conjugated polyclonal anti-SA antibody) was carry out on boiled denatured total cell fraction (TCF) of induced transformants (E. coli BL21(DE3)/pET-26b(+) induced with 0.5 mM IPTG at 37 ˚C) as preliminary confirmation of expression and then on periplasmic fraction of induced host (0.2 mM IPTG at 30 ˚C) to evaluate the formation of soluble tetramers in periplasmic space of the bacteria. The level of periplasmically/secretory expressed tetramer was also investigated in three different temperatures (37 ˚C, 30 ˚C and 20 ˚C) and IPTG concentrations (0.1 mM, 0.2 mM and 0.5 mM) by WB of bacterial periplasmic samples as well as trichloroacetic acid (TCA)-precipitated proteins of culture supernatant.
  • Results: Preliminary analysis of TCF samples of three different transformants showed the strong expression of monomers (~ 42 kDa). WB analysis on periplasm of induced hosts (30 ˚C) disclosed the expression of all forms of the fusion with different intensities (monomer > dimer > tetramer > trimer). The optimization results revealed that the expression level is conversely proportional to temperature and IPTG concentration so that the highest level of expression was related to 20 ˚C/0.1 mM IPTG condition. We surprisingly found that significant amount of the fusion is secreted to the culture medium in 30 ˚C/0.2 mM IPTG condition mostly in tetrameric form (~ 170 kDa).
  • Conclusion: We efficiently expressed soluble tetrameric form of novel scFv-SA molecule in periplasm of E. coli with no observable toxicity and no need to biotin supplementation. Furthermore, we discovered an optimum circumstance in which a large amount of the tetramer species is released to the culture medium. The condition could be used for direct purification of (scFv-SA)4 from culture supernatant using different chromatographic methods.
  • Keywords: single chain antibody; streptavidin; bispecific; periplasm; optimization.