Expression and characterization of the preS1 peptide of hepatitis B surface antigen in Escherichia coli

Abstract

The infectious particles of hepatitis B virus (HBV) contain 3 related surface antigens, i.e., small, medium, and large, all of which are encoded by one large open reading frame with multiple initiation codons. The large surface antigen (L-Ag) contains preS1, preS2, and S regions while both the middle and small surface antigens lack preS1. Several lines of evidence suggested that the preS1 region is involved in the binding of HBV to human hepatocytes as shown by its binding to HepG2 cells and isolated human hepatocyte membranes. To obtain large quantity of preS1 peptide, an expression vector was constructed containing a lac promoter, the 5' half of the beta-galactosidase gene, the Factor Xa tetrapeptide recognition sequence, and the coding region of preS1 plus preS2. This recombinant plasmid constitutively produced high concentration of a fusion protein in inclusion bodies in Escherichia coli. When the fusion protein was treated with Factor Xa, a peptide consisting of the N-terminal 91 amino acids of the preS1 region was released. This preS1 fragment purified by anion exchange chromatography was able to bind specifically to the isolated plasma membranes from human liver. Hence, this recombinant preS1 peptide can be used to identify and isolate hepatocyte receptors for HBV.

Purification and Structural Analysis of the Hepatitis B Virus PreS1 Expressed from Escherichia coli

“…However, purification of intact preS1 has not been successful due to the susceptibility of the polypeptide to degradation during and after its expression in soluble form in bacteria (18,19). Otherwise, the preS1 was expressed as a fusion protein with ␤-galactosidase in insoluble form in bacteria, which needed additional refolding step (20). In this study, we first successfully purified the intact preS1 to near homogeneity from the expressed GST-preS1 fusion protein by a single purification step followed by the immediate lyophilization (Fig.…”

Cloning, Expression, and Purification of Histidine-Tagged preS Domains of Hepatitis B Virus

“…Although the fusion proteins were obtained in fairly good amounts, attempts to separate the preS domain were unsuccessful. Thus, when ␤-galactosidase-preS was treated with factor Xa, a peptide consisting of the first 91 amino acids of the preS1 region was released, but the proteolytic treatment also removed the complete preS2 sequence (36). On the other hand, the full-length preS protein, subtype ayw, was successfully expressed in E. coli as a nonfusion protein using a pET-3a plasmid (24).…”

“…The complete preS domains have been expressed previously in E. coli as fusion proteins at the carboxyterminus of either the maltose binding protein (35) or the ␤-galactosidase (36). Although the fusion proteins were obtained in fairly good amounts, attempts to separate the preS domain were unsuccessful.…”

Delivery of chimeric hepatitis B core particles into liver cells

“…2004). Overexpression of the preS‐related proteins had produced insoluble proteins that were accumulated in inclusion bodies (Lin et al. 1991; Núñez et al.…”