Expression of pre-S2 region of hepatitis B surface antigen in Escherichia coli
Corresponding Author
M. W. Yu Ph.D.
Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892===Search for more papers by this authorJ. W.-K. Shih
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorH. Bursztyn-Pettegrew
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorN. E. Byars
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorA. C. Allison
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorH. W. Chan
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorCorresponding Author
M. W. Yu Ph.D.
Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Division of Blood and Blood Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892===Search for more papers by this authorJ. W.-K. Shih
Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorH. Bursztyn-Pettegrew
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorN. E. Byars
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorA. C. Allison
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorH. W. Chan
Department of Molecular Biology, Syntex Research, Palo Alto, California
Search for more papers by this authorAbstract
We constructed a recombinant plasmid that can express the entire pre-S2 sequence of hepatitis B surface antigen (HBsAg) as a fusion protein in E. coli. The hybrid protein, which comprises the bacterial TrpLE protein and the pre-S2 sequence, was the prominent protein that was found in cell extracts. As determined by immune blot analysis, this protein reacted with human HBV convalescent sera, as well as with sera from animals immunized with either purified HBsAg or isolated polypeptides containing pre-S2. It bound specifically to 125I-polymerized human albumin cross-linked with glutaraldehyde but not to 125I-monomeric human albumin. A novel adjuvant formulation was used in place of Freund's adjuvant to immunize guinea pigs with the recombinant product. The antisera obtained from serial bleedings were found to react with HBsAg of both d and y subtypes. These antisera were also shown to react solely with HBsAg polypeptides which contain the pre-S2 sequence and to block the binding of HBsAg to solid-phase polymerized human albumin.
References
- Bradford M (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248–254.
- Boyer HW, Roulland-Dussoix D (1969): A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology 41: 459–472.
- Byars NE, Allison AC (1987): Adjuvant formulation for use in vaccines to elicit both cell mediated and humoral immunity. Vaccine 5: 223–228.
- Caruthers MH, Beaucage SL, Bedker C, Efcavitch JW, Fisher EF, Galluppi G, Goldman R, deHaseth P, Matteucci M, McBride L, Stabinsky Y (1983): Deoxyoligonucleotide synthesis via the phosphoramidite method. In TS Papas, M Rosenberg, JG Chirkjian (eds): “ Gene Amplification and Analysis.” New York: Elsevier, Vol 3, pp 1–26.
- Charbit A, Sobczak E, Michel M-L, Molia A, Tiollais P, Hofnung M (1987): Presentation of two epitopes of the pre S2 region of hepatitis B virus on live recombinant bacteria. Journal of Immunology 139: 1658–1664.
- Edman JC, Hallewell RA, Valenzuela P, Goodman HM, Rutter WJ (1981): Synthesis of hepatitis B surface and core antigens in E. coli. Nature 291: 503–506.
- Gerin JL, Faust RM, Holland PV (1975): Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits. Journal of Immunology 115: 100–105.
- Heermann KH, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich WH (1984): Large surface proteins of hepatitis B virus containing the pre-s sequence. Journal of Virology 52: 396–402.
- Hellström U, Sylvan S, Kuhns M, Sarin V (1986): Absence of pre-S2 antibodies in natural hepatitis B virus infection. Lancet 2: 889–893.
- Imai M, Yanase Y, Nojiri T, Miyakawa Y, Mayumi M (1979): A receptor for polymerized human and chimpanzee albumins on hepatitis B virus particles co-occurring with HBeAg. Gastroenterology 76: 242–247.
- Itoh Y, Takai E, Ohnuma H, Kitajima K, Tsuda F, Machida A, Mishiro S, Nakamura T, Miyakawa Y, Mayumi M (1986): A synthetic peptide vaccine involving the product of the pre S2 region of hepatitis Bvirus DNA: Protective efficacy in chimpanzees. Proceedings of the National Academy of Sciences USA 83: 9174–9178.
- Koff RS (1986): Comment: Polymerized human albumin binding with hepatitis B surface antigen. Gastroenterology 90: 2031.
- Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
- Lenkei R, Onica D, Ghetie V (1977): Receptors for polymerized albumin on liver cells. Experientia 33: 1046–1047.
- Machida A, Kishimoto S, Ohmma H, Miyamoto H, Baba K, Oda K, Nakamura T, Miyakawa Y, Mayumi M (1983): A hepatitis B surface antigen polypeptide (P31) with the receptor for polymerized human albumin as well as chimapanzee albumins. Gastroenterology 85: 268–274.
- Machida A, Kishimoto S, Ohnuma H, Baba K, Ito Y, Miyamoto H, Funatsu G, Oda K, Usuda S, Togami S, Nakamura T, Miyakawa Y, Mayumi M (1984): A polypeptide containing 55 amino acid residues coded by the pre-S region of hepatitis B virus deoxyribonucleic acid bears the receptor for polymerized human as well as chimpanzee albumins. Gastroenterology 86: 910–918.
- Maxam AM, Gilbert W (1980): Sequencing end-labeled DNA with base specific chemical cleavages. In L Grossman, K Moldave (eds): “ Methods in Enzymology.” New York: Academic Press, Vol 65 (part 1), pp 499–560.
- Milich DR (1987): Immunological response to pre-S antigens of the hepatitis B virus. Viral Immunology 1: 83–96.
- Milich DR, McNamara MK, McLachlan A, Thornton GB, Chisari FV (1985a): Distinct H2-linked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface antigen polypeptide allows circumvention of nonresponsiveness to the S region. Proceedings of the National Academy of Sciences USA 82: 8168–8172.
- Milich DR, Thornton GB, Neurath AR, Kent SBH, Michel M-L. Tiollais P, Chisari FV (1985b): Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen. Science 228: 1195–1199.
- Neurath AR, Kent SBH, Strick N (1984): Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus. Science 224: 392–395.
- Neurath AR, Kent SBH, Strick N, Taylor P, Stevens CE (1985): Hepatitis B virus contains pre-S gene-encoded domains. Nature 315: 154–156.
- Neurath AR, Kent SBH, Parker K, Prince AM, Strick N, Brotman B, Sproul P (1986a): Antibodies to a synthetic peptide from the preS 120–145 region of the hepatitis B virus envelope are virus-neutralizing. Vaccine 4: 35–37.
- Neurath AR, Kent SBH, Strick N, Parker K (1986b): Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 46: 429–436.
- Nichols BP, Yanofsky C (1983): Plasmids containing the trp promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes. In R Wu, L Grossman, K Moldave (eds): “ Methods in Enzymology.” New York: Academic Press, Vol 101 (part C), pp 155–164.
- Okamoto H, Usuda S, Imai M, Tachibana K, Tanaka E, Kumakura T, Itabashi M, Takai E, Tsuda F, Nakamura T, Miyakawa Y, Mayumi M (1986): Antibody to the receptor for polymerized human serum albumin in acute and persistent infection with hepatitis B virus. Hepatology 6: 354–359.
- Pasek M, Goto T, Gilbert W, Zink B, Schaller H, Mackay P, Leadbetter G, Murray K (1979): Hepatitis B virus genes and their expression in E. coli. Nature 282: 575–579.
- Peterson DL (1981): Isolation and characterization of the major protein and glycoprotein of hepatitis B surface antigen. Journal of Biological Chemistry 256: 6975–6983.
- Petit M-A, Maillard P, Capel F, Pillot J (1986): Immunochemical structure of the hepatitis B surface antigen vaccine—II. Analysis of antibody responses in human sera against the envelope proteins. Molecular Immunology 23: 511–523.
- Pontisso P, Schiavon E, Fraiese A, Pornaro E, Realdi G, Alberti A (1986): Antibody to the hepatitis B virus receptor for polymerized albumin in acute infection and in hepatitis B vaccine recipients. Journal of Hepatology 3: 393–398.
- Shih JW-K, Tan PL, Gerin JL (1978): Antigenicity of the major polypeptides of hepatitis B surface antigen (HBsAg). Journal of Immunology 120: 520–525.
- Stibbe W, Gerlich WH (1983): Structural relationships between minor and major proteins of hepatitis B surface antigen. Journal of Virology 46: 626–628.
- Thung SN, Gerber MA (1983): Albumin binding sites of human hepatocytes. Liver 3: 290–294.
- Thung SN, Gerber MA (1984): Polyalbumin receptors: Their role in the attachment of hepatitis B virus to hepatocytes. Seminars in Liver Disease 4: 69–75.
- Towbin H, Stachelin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: Procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76: 4350–4354.
- Trevisan A, Gudat F, Guggenheim R, Krey G, Dürmüller U, Lüond G, Düggelin M, Landmann J, Tondelli P, Bianchi L (1982): Demonstration of albumin receptors on isolated human hepatocytes by light and scanning electron microscopy. Hepatology 2: 832–835.
- Vyas GN, Roa KR, Ibrahim AB (1972): Australia antigen (hepatitis B antigen): A conformational antigen dependent on disulfide bonds. Science 178: 1300–1301.
- Yu MW, Finlayson JS, Shih JW-K (1985): Interaction between various polymerized human albumins and hepatitis B surface antigen. Journal of Virology 55: 736–743.
- Yu MW, Mason BL, Finlayson JS, Shih JW-K (1988): Antipolymerized human albumin and its inhibition of the interaction between polymerized human albumin and HBsAg. In AJ Zuckerman (ed): “ Viral Hepatitis and Liver Disease.” New York: Alan R. Liss, Inc., pp 694–696.
- Zhang W, Shih JW-K, Alter HJ (1985): Constituent hepatitis B surface antigen polypeptides of hepatitis B virus infected patients. Abstract, 1985 Annual Meeting for American Society of Microbiology.
Citing Literature
