Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli

Abstract

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 maxiceils. 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 pl, 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. paUidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.

Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog

“…Whether the 20-kDa protein is expressed in T. pallidum is unknown. A similar result was observed with the recombinant 39-kDa basic membrane protein of T. pallidum (14). Complementation of an ompA mutation with tpn5O.…”

Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity

“…A characteristic feature of the 47-kDa antigen produced either by T. pallidum or by E. coli recombinant clones and analyzed by SDS-PAGE is the frequent presence of a 48-kDa component that reacts with specific polyclonal and monoclonal antibodies (4,11,20,22). It has been suggested that the 48and 47-kDa doublet seen in this and other studies (4, 11,20,22) is the result of inefficient (8) and/or incomplete processing of the 47-kDa antigen. Although it is considered unlikely, it was important to investigate this, particularly in light of the absence of demonstrable processing in E. coli.…”

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum

“…Analyses of the genetics of flagellar expression in other bacteria have proved useful in understanding the genetic organization and control of complex operons (25). Furthermore, analysis of the upstream DNA may provide additional insights into our emerging understanding of the processing of treponemal antigens expressed in E. coli (12,21). In concert with attempts to achieve continuous in vitro cultivation of T. pallidum, the ultimate goal of these studies will be to use the derived genetic information along with mutational analyses to elucidate the relationships between motility and virulence, as has been accomplished with other bacterial pathogens (15).…”