Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein.
A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties.
Jones
H M
HM
Francis I. Proctor Foundation and the Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA.
Kubo
A
A
Stephens
R S
RS
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods; D015151:Immunoblotting; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D018272:Porins / Q000502:physiology; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008081:Liposomes; D018272:Porins; C071677:omp1 protein, Chlamydia trachomatis; C078167:OMPA outer membrane proteins; D004262:DNA Restriction Enzymes