TY - JOUR
T1 - Expression, purification, and functional analysis of an antigen-targeting fusion protein composed of CD40 ligand and the C-terminal fragment of ovalbumin
AU - Shi, Yunnuo
AU - Halperin, Scott A.
AU - Lee, Song F.
N1 - Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2018/2
Y1 - 2018/2
N2 - 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.
AB - 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.
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U2 - 10.1016/j.pep.2017.09.015
DO - 10.1016/j.pep.2017.09.015
M3 - Article
C2 - 28974444
AN - SCOPUS:85030656458
SN - 1046-5928
VL - 142
SP - 37
EP - 44
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -