title: Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli.
abstract: The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli. A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, NY 14642.
authors.forename: C
authors.initials: C
authors.lastname: Dascher
authors.forename: D
authors.initials: D
authors.lastname: Roll
authors.forename: P M
authors.initials: PM
authors.lastname: Bavoil
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D018833:Chaperonins; D002691:Chlamydophila psittaci / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D011498:Protein Precursors; D011994:Recombinant Proteins; D018833:Chaperonins