Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein

Abstract

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polypro tein 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 lb 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 sin gle 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.

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients

“…We designed and expressed ARFP starting at the ?1 position of codon 2 of HCV-4a so that any core?1 protein that may be produced by downstream internal translation initiation would be in frame with the expressed ARFP. A similar approach was used for HCV genotypes 1b and 3, where the stretch of 10 A's was interrupted and the cloned ARFP was designed to start at the ?1 position of codon 8 and 2, respectively [33,34]. Similar to our results, the latter investigators found a significant number of HCV-infected patients who had circulating antibodies against the recombinant ARFP [33,34].…”

“…It could be explained that the additional 9 amino acids in ARFP 2 may play a stabilizing role in the structure of ARFP and its proper folding [29]. Although an ARFP protein lacking these 9 codons was successfully expressed from genotype 1b [34], it was a larger protein composed of 153 amino acids, whereas ARFP 11 has only 115 amino acids.…”

“…Moreover, it has been shown that ARFP can also be recognized by memory T cells from HCV-infected patients [32,44]. Expression of different recombinant forms of ARFP in E. coli was done in previous studies including full-length and truncated forms from HCV genotypes 1a, 1b,1c, 2 and 3 [29][30][31][32][33][34]. However, no information is available on the expression of any form of ARFP from genotype 4a.…”

“…6 Alignment results (A) and phylogenetic tree (B) for ARFP 2 of HCV-4a expressed in this study. ARFP 2 was compared with three previously E. coliexpressed ARFP proteins derived from other HCV genotypes; HCV-1b [34], HCV1c [31] and HCV-3 [33]. The height of columns in the ''Majority'' part of the alignment figure reflects the degree of matching between the four aligned sequences In the present study, a competitive ELISA technique [40] was employed for detecting the ARFP antigen in serum using murine antibody raised against ARFP.…”

“…This ARFP/DF protein was described by Boulant and coworkers [28] for HCV-1b as a protein that consisted of two separate frameshift events: a ?1 frameshift at codon 42 followed by rephasing through a -1 frameshift at position 144 of the core. Recombinant expression of different forms of ARFP in E. coli was done in previous studies, including fulllength or truncated forms of HCV genotypes 1a, 1b,1c, 2 and 3 [29][30][31][32][33][34]. However, no information is available on the expression of any form of ARFP from genotype 4.…”