Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli.
To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (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 fragments 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 region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3-10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease 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 either the first or the third transmembrane domain of the major HBsAg. 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 longer stretch of or a higher ratio of hydrophobic aa had a lower production in E. coli.
Sheu
S Y
SY
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, ROC.
Lo
S J
SJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D016415:Sequence Alignment; D017384:Sequence Deletion
D000596:Amino Acids; D006514:Hepatitis B Surface Antigens; D011993:Recombinant Fusion Proteins