Cloning, Expression of Anti-PDL1/ Anti-CTLA-4  diabody Using Bacterial PelB Leader Sequence  in E.coli BL21 strain

Fathollahi, Yaghoub; Dabirmanesh, Bahareh; Khajeh, Khosrow; Khodakarami, Atefeh

Cloning, Expression of Anti-PDL1/ Anti-CTLA-4 diabody Using Bacterial PelB Leader Sequence in E.coli BL21 strain

Document Type : Original Research

Authors

Yaghoub Fathollahi

Bahareh Dabirmanesh

Khosrow Khajeh

Atefeh Khodakarami

Tarbiat modares university

Abstract

Immune checkpoints are molecules that regulators the immune system. However, some tumor cells can express the ligands of immune checkpoints to escape from antitumor immune responses. Some agents, such as antibodies, can inhibit these checkpoints that prevent the immune system from targeting and killing cancer cells. The aim of this study was to express a novel bispecific diabody in periplasmic space of E.coli for simultaneous targeting of two immune checkpoints, cytotoxic T‑lymphocyte‑associated protein 4 (CTLA‑4) and programmed death- ligand 1 (PD‑L1).

The bispecific diabody was constructed based on the variable regions gene of anti PD-L1 and anti CTLA‑4 antibodies. The optimum codon for expression in E. coli was chemically synthesized and subcloned in pET21 expression plasmid. After transformation, the effect of cultivation conditions on periplasmic expression of the protein in E. coli BL21(DE3) was evaluated. Then, the bispecific diabody was purified .

Expression of diabody with a molecular weight of 55 kDa was verified by Sodium dodecyl sulfate‑polyacrylamide gel electrophoresis and western blotting analysis. The best condition for soluble periplasmic expression was obtained to be incubation with 0.5 mM isopropyl β‑D‑1‑thiogalactopyranoside at 23°C. The protein was successfully purified using affinity chromatography with a final yield of 0.4 mg/L. The affinity of the purified protein interaction were checked by ELISA.

Recombinant Diabody protein was cloned, expressed, and purified in a bacterial system and Diabody Interaction with PDL-1 receptor conformed by Cell-Elisa.

Keywords

Immune checkpoints

Recombinant Diabody

CTLA 4

PD-L1

Subjects

Molecular biotechnology

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