High level expression of hepatitis B virus preS1 peptide in Escherichia coli.
PreS1 region gene fragment encoding the N-terminal 56 amino acid (aa) of hepatitis B virus (HBV, adr subtype), which encodes B- and T-cell epitopes and an hepatocyte receptor binding site, was synthesized by PCR and fused to the 3'-end of MalE gene encoding maltose-binding protein (MBP) to yield expression plasmid pMalpreS1-56. The plasmid was introduced into Escherichia coli DH5 alpha and expressed at 37 degrees C under the control of inducible tac promoter. The resulting fusion protein was highly expressed in a soluble form, about 40% of total cellular proteins, but it bound only partially to an amylose column. Therefore, the soluble preS1 fusion protein was purified to near homogeneity by two passages of anion-exchange chromatography followed by gel filtration. The yield of the fusion protein was 70 mg per 1 culture that had been induced by IPTG for 6 h. The purified fusion protein was specifically cleaved by a Factor Xa digestion to release the preS1 peptide, which was then purified by gel filtration to homogeneity. The purity, integrity, antigenicity and immunogenicity of the purified preS1 peptide was confirmed by glycerol-SDS-PAGE, Western analysis, N-terminal amino acid sequencing and an indirect ELISA.
Rhyum
S B
SB
Protein Engineering Research Group, Genetic Engineering Research Institute, KIST, Taejon, South Korea.
Jin
B R
BR
Park
H R
HR
Hong
H J
HJ
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002850:Chromatography, Gel; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D006514:Hepatitis B Surface Antigens / Q000737:chemistry / Q000235:genetics*; D006515:Hepatitis B virus / Q000737:chemistry / Q000235:genetics*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014759:Viral Envelope Proteins
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D006514:Hepatitis B Surface Antigens; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C461599:maltose transport system, E coli; C050806:presurface protein 1, hepatitis B surface antigen; D015951:Factor Xa