Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12

Embden, J D van; Donk, H J van der; Eijk, Roel van; Heide, H. G. van der; Jong, James A. De; Olderen, M. F. Van; Osterhaus, Albert; Schouls, Leo M.

doi:10.1128/iai.42.1.187-196.1983

Cited by 82 publications

(65 citation statements)

References 24 publications

(14 reference statements)

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“…The expression of cloned treponemal genes in recombinant Escherichia coli has been previously reported by several laboratories (13,16,21,22) and presents a means by which purified T. pallidum molecules could become available for studies of experimental biology and pathogenesis of treponemal infection. The biological significance of the cloned antigens and their correspondence to native T. pallidum polypeptides have not yet been established.…”

mentioning

confidence: 97%

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Fehniger¹,

Walfield²,

Cunningham³

et al. 1984

Infect Immun

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A cloned Treponema pallidum antigen, designated 4D, was purified from Eschenichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190and 90-kd forms of 4D were stable at 68°C but converted to 19and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.

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mentioning

confidence: 97%

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Fehniger¹,

Walfield²,

Cunningham³

et al. 1984

Infect Immun

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“…An antigenic polypeptide, approximating in M r to P3, has been particularly effective in eliciting monoclonal antibodies in mice. Immunization with live treponemes led to isolation of hybridomas reactive with a 46-kDa polypeptide of T. pallidum, and in one instance also with a cloned 44-kDa polypeptide which may thus have been a fragment of the 46 kDa; M r markers used or their values were not given in this study [16]. A cloned antigen of 44 kDa relative to ovalbumin at 46 kDa [19] was strongly expressed and apparently secreted in Escherichia coli, and it was suggested this might also be a fragment of the immunodominant 47-48-kDa polypeptide reported elsewhere [10].…”

Section: Outer Membrane-associated Polypeptide P3mentioning

confidence: 94%

“…Polypeptides approximating to the M r assigned to P3 (47000) feature prominently in the literature [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] as immunodominant antigens and as the target antigen of several reported examples of monoclonal antibodies. Genes encoding polypeptides approximating to this M r value have also been cloned [16,18,19]. We have noted that in some of our profiles of whole treponeme preparations (unpublished), as well as in most of those shown in Fig.…”

Section: S Ds-pa Ge Analysis Of Treponemal Polypeptidesmentioning

confidence: 99%

“…Several groups have now reported the successful cloning in E. coli of genes from T. pallidum encoding antigens reactive with human or animal syphilitic sera [16,18,19,24,46,47]. Polypeptides of 46, 43, 38, 24, 23, 20 and 18.5 kDa were revealed by reaction with human secondary syphilitic serum [18], and were said to correspond with antigenic polypeptides of T. pallidum described subsequently by Hanff and colleagues [9,11,12].…”

Section: Treponemal Antigen Gene Cloningmentioning

confidence: 99%

“…Thus, the cloned 48-kDa polypeptide might have been identical with P3. We have already discussed the cloned polypeptides of 46 [16] and 44 kDa [19], which might be fragments of P3. If all these cloned antigens are P3-related it appears that this antigen is particularly easy to clone, possibly as a result of multiple gene copies or promoters which function strongly in E. coil The nature of most other cloned antigens is less easy to assess.…”

Section: Treponemal Antigen Gene Cloningmentioning

confidence: 99%

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Molecular and immunochemical analysis ofTreponema pallidum

Penn

Bailey

1986

FEMS Microbiology Letters

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Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent ‘landmark’ polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane‐associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa.

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Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results from different laboratories

Norris¹,

Alderete

Axelsen

et al. 1987

Electrophoresis

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Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfatepolyacrylamide gel electrophoresis results from different laboratoriesAs the first step in a cooperative effort to standardize the identification of the polypeptides of Treponerna pallidum subsp. pallidurn, the sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) results obtained in 16 laboratories were compared. Although it was possible to correlate the positions of 16 ofthe major polypeptide bands, the cross-identification of many of the polypeptides was ambiguous, particularly in the low molecular weight range. Two-dimensional electrophoresis provided an improved means of separating and characterizing T.pallidum polypeptides as isolated molecular species. An approach to the unambiguous identification of treponemal polypeptides was outlined which will utilize two-dimensional electrophoresis in combination with specific properties attributable to individual proteins, including reactivity with monoclonal antibodies or monospecific antisera, biochemical and structural properties, and sequence information. To demonstrate the feasibility of this approach, two-dimensional electrophoresis in conjunction with immunoperoxidase staining was used to specifically identify three cloned T.pal1idum proteins. Abbreviations: CIE, crossed immunoelectrophoresis; 2DE, two-dimensional gel electrophoresis; IRS, infected rabbit serum; 2-ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; RIP, radioimmunoprecipitation: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; T, Tween 20 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0173-0835/87/0202-0077 %02.50/0 S. J. Norris et al. Electrophoresis 1987,8, 77-92 Figure 16. Reactivity of rabbit antiserum directed against cloned antigenTmpC (1301, seeFig. 6)with the 2DEpatternof T.paNidum.Theprocedure wasas described in Fig. 15. (A) Reaction with the TmpC-specific antiserum. (B) Antigenic profile following IRS counterstaining. The antiserum reacted specifically with a single band on the SDS-PAGE pattern (far left) and a single spot at the acid end of the 2DE pattern.Figure 17. Localization of cloned antigen TpD (1301, see Fig. 6) in the 2DE profile of T.pallidum, utilizing a specific rabbit antiserum prepared by affinity chromatography. (A) Reactivity of TpD-specific antiserum. Due to prolonged incubation with the color reagent, background reactivities of the antiserum to TmpC and the major 61 polypeptide were also detectable in this case. (B) Antigenic profile following IRS counterstaining. The positions of TpD and TmpC in the 2D and single dimension SDS-PAGE profiles are indicated by the arrows.

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Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12

Cited by 82 publications

References 24 publications

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Molecular and immunochemical analysis ofTreponema pallidum

Identity of Treponema pallidum subsp. pallidum polypeptides: Correlation of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results from different laboratories

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