A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start ftom at least two different treponemal promnoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coil, we concluded that a similar proteolytic processing takes place in both E. coli and T.paflidum6 Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent thlah tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front bf the structural genes. The nucleotide sequence of the T. pallidum tmpA geite was established. This is the first T. paUidim gene sequenced. Codon usage and the nature of transcriptional and translational signals ate discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a s;gnal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having I-galactosidase enzyme activity as well as TmpA epitopes. The enzymelinked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage A were used. In this case the protein produced was a sandwich protein consisting of 21 aminio acids of the A cro protein, 204 amnino acids of the T. pallidam TmpA protein, and 1,020 amino lcids of the E. coli X-galictosidase. The potedtial use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed. Treponema pallidum is the causative agent of syphilis. Because the microorganism cannot be grown in vitro, little is known about the factors that contribute to its virulence and the immunologic response duting a syphilitic infection. For this reason various groups have cloned genes of T. pallidum that encode for immunogenic proteins (17, 24, 27, 30). These recent studies have shown that T. pallidum DNA can be expressed well in Escherichia coli, and treponemal transcriptional and translational signals appear to be recognized by the E. coli machinery for protein synthesis. Recently, we screened a cosmid library with T. pallidum DNA inserts for the production of T. pallidum antigens (27). Among the 800 clones scteened, one particular phenotype was found most frequently. Of 16 antigen-producing clones,
A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.
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