Generation of a fluorescent oncoprotein in soluble form and its delivery into mammalian cells.

BACKGROUND: Protein fusion technology was widely used to improve expression, purification and solubility of the recombinant proteins expressed in E. coli for in vitro/in vivo delivery. METHODS: We developed a method for successful expression of soluble +36GFP-A2-E7 protein in E. coli and its effective delivery into mammalian cells. At first, the plasmid harboring +36GFP-A2-E7 was transformed into E. coli Rosetta competent cells. Then, the recombinant protein fused to histidine tag was expressed and purified using affinity chromatography. Different conditions such as inducer dose, time and temperature of induction, pH and urea concentration were evaluated. Finally, the delivery of the recombinant protein was detected in HEK-293T cells using fluorescent microscopy and flow cytometry. RESULTS: Our data showed that the expressed protein formed inclusion bodies at 37 °C and 3 h post-induction. The soluble protein was generated using 0.5 mM IPTG and growth at 16 °C for 20 h, and purified by low concentrations of urea and 200 mM imidazole. The soluble fraction of +36GFP-A2-E7 protein could significantly represent higher fluorescent property and stronger delivery into mammalian cells compared to the insoluble form. CONCLUSION: Generally, soluble form of fusion protein retained its biological activity and could directly penetrate into the cells without the fusion tags (Tab. 1, Fig. 7, Ref. 23).

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.