Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.
A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.
D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D010957:Plasmids; D014210:Treponema pallidum / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008565:Membrane Proteins; D004247:DNA