Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein.
Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called "ARFP/F", or "core+1", protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11-161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the 1b-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran 16535446, Iran.
F
F
Baghbani-arani
F
F
Roohvand
M R
MR
Aghasadeghi
A
A
Eidi
S
S
Amini
F
F
Motevalli
S M
SM
Sadat
A
A
Memarnejadian
G
G
Khalili
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D018985:Epitopes, B-Lymphocyte / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli; D015870:Gene Expression; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus