Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin

Yunnuo Shi, Scott A. Halperin, Song F. Lee

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

Delivering antigen via molecules specifically targeting receptors on the surface of antigen-presenting cells is a strategy to improve immune responses. In this study, an antigen-targeting fusion protein (OVA-CD40LS) composed of the C-terminal fragment of ovalbumin and the extracellular domain of mouse CD40 ligand was constructed by genetic fusion. The OVA-CD40LS and the control OVA (rOVA) genes were cloned in Escherichia coli and over-expressed as insoluble proteins. The rOVA protein was purified from the insoluble fraction of E. coli cell lysate by nickel affinity chromatography and refolded by step-wise dialysis to give a yield of 11.8 mg/L of culture. The OVA-CD40LS was purified by a ‘two-round’ nickel affinity and on-column protein-refolding chromatography. The yield was 528 μg/L of culture. The purified OVA-CD40LS, but not the rOVA, was able to simulate the production of pro-inflammatory cytokines and up-regulate cell surface marker proteins in mouse bone marrow-derived dendritic cells. The purified OVA-CD40LS elicited a robust immune response when injected submucosally in the oral cavity of mice. Collectively, the results indicate that the OVA-CD40LS fusion protein was biologically active, functioning as an antigen-targeting protein.

Original languageEnglish
Pages (from-to)37-44
Number of pages8
JournalProtein Expression and Purification
Volume142
DOIs
Publication statusPublished - Feb 2018

Bibliographical note

Publisher Copyright:
© 2017 Elsevier Inc.

ASJC Scopus Subject Areas

  • Biotechnology

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