DELETION OR ALTERATION OF HYDROPHOBIC AMINO-ACIDS AT THE FIRST AND THE 3RD TRANSMEMBRANE DOMAINS OF HEPATITIS-B SURFACE-ANTIGEN ENHANCES ITS PRODUCTION IN ESCHERICHIA-COLI

To investigate the failure of high-level production of hepatitis B vir al (HBV) surface antigen (HBsAg), including three authentic forms, lar ge (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S- transferase-encoding gene (GST) to study HBsAg production. Different f ragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S regi on (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PA GE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion prote ins, in contrast to those cells harboring plasmids without the S regio n (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3-10% of the total cellular protein. Using an immunoblot method to screen HB sAg production in cells which harbored plasmids derived from exonuclea se BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding e ither the first or the third transmembrane domain of the major HBsBg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 aa among 15 clones suggested that the fusion protein ended with a l onger stretch of or a higher ratio of hydrophobic aa had a lower produ ction in E. coli.