As described in the previous annual report, the more recent preparations of our (wild type) recombinant preS1 peptide (rpreS1) contained two peptides which are only separable by SDS-PAGE. When this rpreS1 preparation was used as a labeled ligand by conjugating with 125 I-Bolton-Hunter reagent,no specific binding to either plasma membranes prepared from human hepatocytes or Hep G2 cells was observed. A sample of this rpreS1 preparation was sent to Dr. Neurath but tested negative in its competition capability in his unique binding system of HBsAg to HepG2 cells. Hence, point mutations were performed and produced a mutant recombinant plasmid which can express a fusion protein containing a 90 amino acid mutant preS1 peptide in E. coli. Upon Factor Xa digestion, this mutant rpreS1 peptide, which contains tyr12 tyr13 rather than a wild type phe12 phe13 and a C-terminal gly90, was released. The mutant rpreS1 has been purified by mono-Q column chromatography, and 18 N-terminal amino acid residues have been confirmed by amino acid sequence analysis. This mutant rpreS1 can be labeled with carrier-free Na 125I by means of the Iodogen method because of the availability of tyrosine residues. Binding studies are in progress.
