Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

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Movaghar Asareh Shirin; Savei Tahereh; ARJMAND SAREH; RANAEI SIADAT SEYED OMID; Fatemi Fateneh; Pourmadadi Mehrab; Shabani Shayeh Javad

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Journal: BioImpacts Year:2022 | Volume:12 | Issue:3 Page(s): 203-210

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Title

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris

Author(s)

Keywords

Ranibizumab

Fab fragment

VEGF-A

Pichia pastoris

Split reporter

eGFP

Abstract

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet agerelated macular degeneration (AMD). Methods: In this study, the heavy and light chains of Ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4° C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the Split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of Ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for Ranibizumab production.

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APA: Copy

Movaghar Asareh, Shirin, Savei, Tahereh, ARJMAND, SAREH, RANAEI SIADAT, SEYED OMID, Fatemi, Fateneh, Pourmadadi, Mehrab, & Shabani Shayeh, Javad. (2022). Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris. BIOIMPACTS, 12(3), 203-210. SID. https://sid.ir/paper/978637/en

Vancouver: Copy

Movaghar Asareh Shirin, Savei Tahereh, ARJMAND SAREH, RANAEI SIADAT SEYED OMID, Fatemi Fateneh, Pourmadadi Mehrab, Shabani Shayeh Javad. Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris. BIOIMPACTS[Internet]. 2022;12(3):203-210. Available from: https://sid.ir/paper/978637/en

IEEE: Copy

Shirin Movaghar Asareh, Tahereh Savei, SAREH ARJMAND, SEYED OMID RANAEI SIADAT, Fateneh Fatemi, Mehrab Pourmadadi, and Javad Shabani Shayeh, “Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris,” BIOIMPACTS, vol. 12, no. 3, pp. 203–210, 2022, [Online]. Available: https://sid.ir/paper/978637/en

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