Cloning, expression, purification and characterization of the hepatitis B viral pre-S, X and RNase H proteins

The hepatitis virus (HBV) adw and ayw subtype preS gene have been cloned down stream of T7 promotor and used to direct the expression of the preS protein in E. coli. To overcome the difficulty in purifying the protein, the preS-polyhistidine fusions were constructed. Proteins were readily purified using nickel chelation chromatography. The expressed his-tagged protein has a molecular weight of 20 kDa for adw preS and 18 kDa for ayw preS. Both proteins are characterized. Circular dichroism spectroscopy revealed that adw preS which was purified under denaturing condition, exhibits no periodic secondary structure; ayw preS which was purified under native condition, exhibits a primary {dollar}beta{dollar}-sheet secondary structure which is the same as non his-tagged protein.; ADW preS was radiolabeled by {dollar}sp{lcub}125{rcub}{dollar}I and its binding to human and rabbit liver cell membranes was studied. It was found in this study that {dollar}sp{lcub}125{rcub}{dollar}I-preS binds specifically to human liver plasma membranes, but that it does not bind to human liver inner membranes or to rabbit liver plasma membranes. Scatchard analysis revealed a single class of binding sites with a Kd = 12 nM and Bmax 0.34 pmol/60 ug protein. The preincubation with polymerized human serum albumin does not increase the preS binding to the human plasma membrane.; The direct and indirect sandwich ELISA method were developed to detect antibody production in serum. The direct sandwich ELISA uses peroxidase-CM-lys-Ni-preS to detect anti-preS antibody. The indirect ELISA uses the peroxidase-anti-human IgG to detect anti-preS antibody. Both methods are specific and sensitive. The anti-preS production pattern was studied in chimpanzee and human HBV infection panel, random acute and chronic samples. It was found that the preS antibody appeared at the early stage of HBV infection (in 2-3 months from virus inoculation); 81% of the acute infection samples have antibody response, 45% of the chronic infection has antibody production but the antibody level was extremely low in chronic patients (OD {dollar}<{dollar} 0.2).; In a second study, the X gene was cloned into the pQE-60 expression vector multiple cloning sites. The X protein of the ayw subtype HBV virus was expressed in E. coli and purified to homogeneity from inclusion bodies. The purified protein has been shown to have the expected N-terminal amino acid sequence by Edman degradation. Immunoblotting studies have demonstrated that the purified X protein binds to rabbit anti-X and HBV infected patients' serum. GC mass spectrometry analysis showed that the protein has a molecular mass of 17.5 kDa, with a small amount of dimeric form.; A sandwich ELISA was developed, using recombinant X protein, to detect anti-X antibodies in human sera. The assay successfully identified 18% positive sera in chronically infected samples, only 6% of acute infected samples were positive.; In the last study, the RNase H domain of the ayw HBV polymerase gene was cloned into pET21a vector. The protein was expressed in E. coli and purified to yield 1-2 mg of protein per liter of culture. The expressed protein has a carboxyterminal polyhistidine tag to facilitate purification. It has been shown to degrade the RNA component of RNA/DNA hybrids, but not the DNA component. The RNase H activity has a basic pH optimum, is active only in the presence of reducing agents, and is dependent on the presence of magnesium.