RESUMO
An E. coli expression system offers a mean for rapid, high yield and economical production of Hepatitis B Virus core (HBc) particles. However, high-level production of HBc particles in bacteria is demanding and optimisation of HBc particle yield from E. coli is required to improve laboratory-scale productivity for further drug delivery applications. Production steps involve bacterial culture, protein isolation, denaturation, purification and finally protein assembly. In this study, we describe a modified E. coli based method for purifying HBc particles and compare the results with those obtained using a conventional purification method. HBc particle morphology was confirmed by Atomic Force Microscopy (AFM). Protein specificity and secondary structure were confirmed by Western Blot and Circular Dichroism (CD), respectively. The modified method produced ~3-fold higher yield and greater purity of wild type HBc particles than the conventional method. Our results demonstrated that the modified method produce a better yield and purity of HBc particles in an E. coli-expression system, which are fully characterised and suitable to be used for drug delivery applications.
Assuntos
Portadores de Fármacos/isolamento & purificação , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Proteínas Recombinantes/metabolismo , Vírion/isolamento & purificação , Western Blotting , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/ultraestrutura , Microscopia de Força Atômica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vírion/genética , Vírion/ultraestruturaRESUMO
BACKGROUND: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues. RESULTS: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells. CONCLUSIONS: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.