Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3′ end in mRNA stability

Ravi Kaul, Matthew J J Duncan, James D Guest, Wanda M. Wenman

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)97-103
Number of pages7
JournalGene
Volume87
Issue number1
DOIs
StatePublished - Mar 1 1990
Externally publishedYes

Fingerprint

RNA Stability
Membrane Proteins
Escherichia coli
Genes
Chlamydia trachomatis
Proteins
Membranes
Epitopes
Amino Acid Sequence
Plasmids
Ethanol
Monoclonal Antibodies
Chlamydia trachomatis omp1 protein
Amino Acids

Keywords

  • epitope
  • gene product
  • leader polypeptide
  • minicells
  • monoclonal antibodies
  • phage λ vectors
  • preprocessed polypeptide
  • Recombinant DNA

ASJC Scopus subject areas

  • Genetics

Cite this

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli : role of the 3′ end in mRNA stability. / Kaul, Ravi; Duncan, Matthew J J; Guest, James D; Wenman, Wanda M.

In: Gene, Vol. 87, No. 1, 01.03.1990, p. 97-103.

Research output: Contribution to journalArticle

@article{10492c0cc5e7436588bcc9e04c842fed,
title = "Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3′ end in mRNA stability",
abstract = "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.",
keywords = "epitope, gene product, leader polypeptide, minicells, monoclonal antibodies, phage λ vectors, preprocessed polypeptide, Recombinant DNA",
author = "Ravi Kaul and Duncan, {Matthew J J} and Guest, {James D} and Wenman, {Wanda M.}",
year = "1990",
month = "3",
day = "1",
doi = "10.1016/0378-1119(90)90499-H",
language = "English",
volume = "87",
pages = "97--103",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1",

}

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 D

AU - Wenman, Wanda M.

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

UR - http://www.scopus.com/inward/record.url?scp=0025303945&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025303945&partnerID=8YFLogxK

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 -