Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system.
We previously reported the complete primary structure of the 37-kilodalton endoflagellar sheath protein (FlaA) of Treponema pallidum. However, we were unable to determine the nucleotide sequence of flaA upstream of amino acid 10. The desired nucleotide sequence was obtained by use of a strategy based upon the polymerase chain reaction and was found to contain a consensus Escherichia coli promoter, a ribosomal binding site, and a 20-amino-acid signal peptide. Expression of FlaA in E. coli was achieved by cloning polymerase chain reaction-derived constructs lacking the native T. pallidum promoter into a temperature-inducible T7 expression system. Pulse-chase and ethanol inhibition analyses of protein processing in E. coli cells and minicells, respectively, indicated that processing of the FlaA precursor was incomplete. Native and recombinant FlaA were identical as assessed by antibody reactivity and sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoretic mobilities. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that FlaA precursor and FlaA were associated with both the cytoplasmic and outer membranes. This is the first report of expression in E. coli of a T. pallidum protein which could not be cloned or expressed with its native promoter. Our data also indicate that information obtained in E. coli regarding the subcellular location of cloned treponemal proteins must be cautiously extrapolated to T. pallidum.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
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RD
Isaacs
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Radolf
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D005407:Flagella / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D014210:Treponema pallidum / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins