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. 1983 Nov;42(2):435–445. doi: 10.1128/iai.42.2.435-445.1983

Cloning and expression of Treponema pallidum (Nichols) antigen genes in Escherichia coli.

M V Norgard, J N Miller
PMCID: PMC264449  PMID: 6358023

Abstract

Hybrid pBR322 plasmid clone banks comprised of more than 125,000 recombinant DNA clones and representing the entire Treponema pallidum Nichols genome were constructed in Escherichia coli K-12 RR1. The two clone banks individually contain over 53,000 and 72,000 recombinant clones. The number average and mass average sizes of the cloned DNA inserts were found to be approximately 12 and 13 kilobase pairs, respectively, indicating the presence of large treponemal DNA inserts in a majority of recombinant clones. To detect E. coli clones synthesizing T. pallidum antigens as hybrid plasmid gene translation products in the clone bank, a simplified, direct, solid-phase radioimmuno-colony blot (RICB) assay was developed employing immunoglobulin G antibody isolated from anti-T. pallidum immune rabbit serum. Clones with positive reactivities in the RICB assay were isolated at frequencies of 0.1 to 0.2%. One isolated RICB-positive clone, designated RICB2-1, produced a very strong signal in the RICB assay and was subsequently found through E. coli cell-free in vitro transcription/translation analysis to encode the synthesis of two gene translation products with apparent molecular weights of 77,000 and 44,000. The 44,000-dalton protein was effectively immunoprecipitated from [35S]methionine-labeled E. coli clone cells by using either immune rabbit serum (preabsorbed with Treponema phagedenis biotype Reiter antigens) or selected human syphilitic serum, whereas the 77,000-dalton protein was never immunoprecipitable by similar methods. Purified plasmid DNA from clone RICB2-1 contained a treponemal DNA insert of 3.70 kilobase pairs, which was of suitable size to code for the 121-dalton (44 + 77) protein. The insert was also flanked on each end by PstI sites and possessed three internal PstI sites with fragment sizes of 2.15, 1.18, 0.20, and 0.17 kilobase pairs. Purified clone RICB2-1 plasmid DNA was capable of transforming recipient E. coli cells to virtually 100% RICB reactivity, thus substantiating the plasmid-encoded characteristic. Further experiments employing various antisera in radioimmunoprecipitation systems utilizing cell-free in vitro synthesized gene translation products from clone RICB2-1 also provided the first evidence that E. coli may be capable of using endogenous T. pallidum DNA promotors for genetic expression. These studies, amplified by the isolation of a potentially significant immunoprecipitable 44,000-dalton recombinant protein antigen, point to the importance of the "cloned antigen gene" approach for the eventual eludication of specific antigens or immunogens operative in the pathogenesis, immunology, and serodiagnosis of T. pallidum infection.

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Selected References

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  1. Alderete J. F., Baseman J. B. Analysis of serum IgG against Treponema pallidum protein antigens in experimentally infected rabbits. Br J Vener Dis. 1981 Oct;57(5):302–308. doi: 10.1136/sti.57.5.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alderete J. F., Baseman J. B. Surface characterization of virulent Treponema pallidum. Infect Immun. 1980 Dec;30(3):814–823. doi: 10.1128/iai.30.3.814-823.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ambler R. P., Scott G. K. Partial amino acid sequence of penicillinase coded by Escherichia coli plasmid R6K. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3732–3736. doi: 10.1073/pnas.75.8.3732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baseman J. B., Hayes E. C. Molecular characterization of receptor binding proteins and immunogens of virulent Treponema pallidum. J Exp Med. 1980 Mar 1;151(3):573–586. doi: 10.1084/jem.151.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bishop N. H., Miller J. N. Humoral immunity in experimental syphilis. I. The demonstration of resistance conferred by passive immunization. J Immunol. 1976 Jul;117(1):191–196. [PubMed] [Google Scholar]
  7. Bishop N. H., Miller J. N. Humoral immunity in experimental syphilis. II. The relationship of neutralizing factors in immune serum to acquired resistance. J Immunol. 1976 Jul;117(1):197–207. [PubMed] [Google Scholar]
  8. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  9. Broome S., Gilbert W. Immunological screening method to detect specific translation products. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2746–2749. doi: 10.1073/pnas.75.6.2746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke L., Carbon J. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome. Cell. 1976 Sep;9(1):91–99. doi: 10.1016/0092-8674(76)90055-6. [DOI] [PubMed] [Google Scholar]
  11. Erlich H. A., Cohen S. N., McDevitt H. O. A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments. Cell. 1978 Apr;13(4):681–689. doi: 10.1016/0092-8674(78)90218-0. [DOI] [PubMed] [Google Scholar]
  12. Fieldsteel A. H., Cox D. L., Moeckli R. A. Cultivation of virulent Treponema pallidum in tissue culture. Infect Immun. 1981 May;32(2):908–915. doi: 10.1128/iai.32.2.908-915.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fieldsteel A. H., Cox D. L., Moeckli R. A. Further studies on replication of virulent Treponema pallidum in tissue cultures of Sf1Ep cells. Infect Immun. 1982 Feb;35(2):449–455. doi: 10.1128/iai.35.2.449-455.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fitzgerald T. J., Johnson R. C., Ritzi D. M. Relationship of Treponema pallidum to acidic mucopolysaccharides. Infect Immun. 1979 Apr;24(1):252–260. doi: 10.1128/iai.24.1.252-260.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fitzgerald T. J., Johnson R. C. Surface mucopolysaccharides of Treponema pallidum. Infect Immun. 1979 Apr;24(1):244–251. doi: 10.1128/iai.24.1.244-251.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fitzgerald T. J., Johnson R. C., Wolff E. T. Mucopolysaccharide material resulting from the interaction of Treponema pallidum (Nichols strain) with cultured mammalian cells. Infect Immun. 1978 Nov;22(2):575–584. doi: 10.1128/iai.22.2.575-584.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fitzgerald T. J. Pathogenesis and immunology of Treponema pallidum. Annu Rev Microbiol. 1981;35:29–54. doi: 10.1146/annurev.mi.35.100181.000333. [DOI] [PubMed] [Google Scholar]
  18. Gulig P. A., McCracken G. H., Jr, Frisch C. F., Johnston K. H., Hansen E. J. Antibody response of infants to cell surface-exposed outer membrane proteins of Haemophilus influenzae type b after systemic Haemophilus disease. Infect Immun. 1982 Jul;37(1):82–88. doi: 10.1128/iai.37.1.82-88.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. HARDY P. H., Jr, NELL E. E. Study of the antigenic structure of Treponema pallidum by specific agglutination. Am J Hyg. 1957 Sep;66(2):160–172. doi: 10.1093/oxfordjournals.aje.a119893. [DOI] [PubMed] [Google Scholar]
  20. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  21. Hanff P. A., Fehniger T. E., Miller J. N., Lovett M. A. Humoral immune response in human syphilis to polypeptides of Treponema pallidum. J Immunol. 1982 Sep;129(3):1287–1291. [PubMed] [Google Scholar]
  22. Hansen E. J., Frisch C. F., Johnston K. H. Detection of antibody-accessible proteins on the cell surface of Haemophilus influenzae type b. Infect Immun. 1981 Sep;33(3):950–953. doi: 10.1128/iai.33.3.950-953.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Henning U., Schwarz H., Chen R. Radioimmunological screening method for specific membrane proteins. Anal Biochem. 1979 Aug;97(1):153–157. doi: 10.1016/0003-2697(79)90339-7. [DOI] [PubMed] [Google Scholar]
  24. Kung H. F., Redfield B., Weissbach H. DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract. J Biol Chem. 1979 Sep 10;254(17):8404–8408. [PubMed] [Google Scholar]
  25. Kung H., Spears C., Weissbach H. Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase. J Biol Chem. 1975 Feb 25;250(4):1556–1562. [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lukehart S. A., Baker-Zander S. A., Gubish E. R., Jr Identification of Treponema pallidum antigens: comparison with a nonpathogenic treponeme. J Immunol. 1982 Aug;129(2):833–838. [PubMed] [Google Scholar]
  28. MAGNUSON H. J., THOMAS E. W., OLANSKY S., KAPLAN B. I., DE MELLO L., CUTLER J. C. Inoculation syphilis in human volunteers. Medicine (Baltimore) 1956 Feb;35(1):33–82. doi: 10.1097/00005792-195602000-00002. [DOI] [PubMed] [Google Scholar]
  29. METZGER M., HARDY P. H., Jr, NELL E. E. Influence of lysozyme upon the treponeme immobilization reaction. Am J Hyg. 1961 Mar;73:236–244. doi: 10.1093/oxfordjournals.aje.a120182. [DOI] [PubMed] [Google Scholar]
  30. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  31. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  32. Metzger M., Michalska E., Podwińska J., Smogór W. Immunogenic properties of the protein component of Treponema pallidum. Br J Vener Dis. 1969 Dec;45(4):299–304. doi: 10.1136/sti.45.4.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miao R., Fieldsteel A. H. Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes. J Bacteriol. 1978 Jan;133(1):101–107. doi: 10.1128/jb.133.1.101-107.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miller J. N. Immunity in experimental syphilis. VI. Successful vaccination of rabbits with Treponema pallidum, Nichols strain, attenuated by -irradiation. J Immunol. 1973 May;110(5):1206–1215. [PubMed] [Google Scholar]
  35. Miller J. N., de Bruijn J. H., Bekker J. H. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri. J Bacteriol. 1966 Feb;91(2):583–587. doi: 10.1128/jb.91.2.583-587.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Morrison-Plummer J., Alderete J. F., Baseman J. B. Enzyme-linked immunosorbent assay for the detection of serum antibody to outer membrane proteins of Treponema pallidum. Br J Vener Dis. 1983 Apr;59(2):75–79. doi: 10.1136/sti.59.2.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Norgard M. V., Emigholz K., Monahan J. J. Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside. J Bacteriol. 1979 Apr;138(1):270–272. doi: 10.1128/jb.138.1.270-272.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Norgard M. V., Keem K., Monahan J. J. Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA. Gene. 1978 Jul;3(4):279–292. doi: 10.1016/0378-1119(78)90038-0. [DOI] [PubMed] [Google Scholar]
  39. Norgard M. V., Miller J. N. Plasmid DNA in Treponema pallidum (Nichols): potential for antibiotic resistance by syphilis bacteria. Science. 1981 Jul 31;213(4507):553–555. doi: 10.1126/science.6264606. [DOI] [PubMed] [Google Scholar]
  40. Norgard M. V. Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase. Anal Biochem. 1981 May 1;113(1):34–42. doi: 10.1016/0003-2697(81)90040-3. [DOI] [PubMed] [Google Scholar]
  41. Norgard M. V., Tocci M. J., Monahan J. J. On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli. J Biol Chem. 1980 Aug 25;255(16):7665–7672. [PubMed] [Google Scholar]
  42. Norris S. J. In vitro cultivation of Treponema pallidum: independent confirmation. Infect Immun. 1982 Apr;36(1):437–439. doi: 10.1128/iai.36.1.437-439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peacock S. L., McIver C. M., Monahan J. J. Transformation of E. coli using homopolymer-linked plasmid chimeras. Biochim Biophys Acta. 1981 Sep 28;655(2):243–250. doi: 10.1016/0005-2787(81)90014-9. [DOI] [PubMed] [Google Scholar]
  44. Raetz C. R. Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2274–2278. doi: 10.1073/pnas.72.6.2274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Robertson S. M., Kettman J. R., Miller J. N., Norgard M. V. Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols). Infect Immun. 1982 Jun;36(3):1076–1085. doi: 10.1128/iai.36.3.1076-1085.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  48. Stamm L. V., Bassford P. J., Jr Cloning and expression of Treponema pallidum protein antigens in Escherichia coli. DNA. 1982;1(4):329–333. doi: 10.1089/dna.1982.1.329. [DOI] [PubMed] [Google Scholar]
  49. Stamm L. V., Folds J. D., Bassford P. J., Jr Expression of Treponema pallidum antigens in Escherichia coli K-12. Infect Immun. 1982 Jun;36(3):1238–1241. doi: 10.1128/iai.36.3.1238-1241.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. TURNER T. B., KLUTH F. C. Protective antibodies in the serum of syphilitic patients. Am J Hyg. 1948 Sep;48(2):173–181. doi: 10.1093/oxfordjournals.aje.a119233. [DOI] [PubMed] [Google Scholar]
  52. TURNER T. B., NELSON R. A., Jr The relationship of treponemal immobilizing antibody to immunity in syphilis. Trans Assoc Am Physicians. 1950;63:112–117. [PubMed] [Google Scholar]
  53. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Titus R. G., Weiser R. S. Experimental syphilis in the rabbit: passive transfer of immunity with immunoglobulin G from immune serum. J Infect Dis. 1979 Dec;140(6):904–913. doi: 10.1093/infdis/140.6.904. [DOI] [PubMed] [Google Scholar]
  55. Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Walfield A. M., Hanff P. A., Lovett M. A. Expression of Treponema pallidum antigens in Escherichia coli. Science. 1982 Apr 30;216(4545):522–523. doi: 10.1126/science.7041257. [DOI] [PubMed] [Google Scholar]
  57. Zeigler J. A., Jones A. M., Jones R. H., Kubica K. M. Demonstration of extracellular material at the surface of pathogenic T. pallidum cells. Br J Vener Dis. 1976 Feb;52(1):1–8. doi: 10.1136/sti.52.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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