TY - JOUR
T1 - Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli
T2 - role of the 3′ end in mRNA stability
AU - Kaul, Ravi
AU - Duncan, Matthew J.J.
AU - Guest, James
AU - Wenman, Wanda M.
N1 - Funding Information:
This work was supported by grants from the Canadian Medical Research Council (MA7951) and University of Alberta Central Research Fund. W.M.W. is a scholar ofthe Alberta Heritage Foundation for Medical Research. We thank I. Clarke, University of Southampton, for providing a MOMP species-specific oligodeoxyribonucleotide; R. Barnes, CDC, Atlanta, for providing monoclonal antibody KG5; R. Meuser for technical help and L. Frost, University of Alberta, for providing plasmid pKO4 and E. coli ED 5363.
PY - 1990/3/1
Y1 - 1990/3/1
N2 - The major outer membrane protein (MOMP)-encoding gene (ompl) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant ompl is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous ompl promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3′ end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed ompl gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.
AB - The major outer membrane protein (MOMP)-encoding gene (ompl) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant ompl is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous ompl promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3′ end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed ompl gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.
KW - epitope
KW - gene product
KW - leader polypeptide
KW - minicells
KW - monoclonal antibodies
KW - phage λ vectors
KW - preprocessed polypeptide
KW - Recombinant DNA
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U2 - 10.1016/0378-1119(90)90499-H
DO - 10.1016/0378-1119(90)90499-H
M3 - Article
C2 - 2139622
AN - SCOPUS:0025303945
VL - 87
SP - 97
EP - 103
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -