The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters

Douglas A. Kuntz, Margaret A. Phillips, Tracey D E Moore, Sydney P. Craig, Kathryn E. Bass, Ching C. Wang

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the λ phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the λ PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5′-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the λ PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the phoA promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.

Original languageEnglish (US)
Pages (from-to)95-104
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume55
Issue number1-2
DOIs
StatePublished - 1992

Fingerprint

Trypanosoma brucei brucei
Ornithine Decarboxylase
Methionine
Enzymes
Molecular Weight
Coliphages
Escherichia coli
Recombinant Proteins
Open Reading Frames
Genes
Plasmids
Messenger RNA

Keywords

  • mRNA structure
  • Ornithine decarboxylase
  • Recombinant expression
  • Trans-splicing
  • Trypanosoma brucei

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters. / Kuntz, Douglas A.; Phillips, Margaret A.; Moore, Tracey D E; Craig, Sydney P.; Bass, Kathryn E.; Wang, Ching C.

In: Molecular and Biochemical Parasitology, Vol. 55, No. 1-2, 1992, p. 95-104.

Research output: Contribution to journalArticle

Kuntz, Douglas A. ; Phillips, Margaret A. ; Moore, Tracey D E ; Craig, Sydney P. ; Bass, Kathryn E. ; Wang, Ching C. / The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters. In: Molecular and Biochemical Parasitology. 1992 ; Vol. 55, No. 1-2. pp. 95-104.
@article{3eb01352cbe54e97b17d25c1e4d7b621,
title = "The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters",
abstract = "Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the λ phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the λ PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5′-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the λ PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the phoA promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.",
keywords = "mRNA structure, Ornithine decarboxylase, Recombinant expression, Trans-splicing, Trypanosoma brucei",
author = "Kuntz, {Douglas A.} and Phillips, {Margaret A.} and Moore, {Tracey D E} and Craig, {Sydney P.} and Bass, {Kathryn E.} and Wang, {Ching C.}",
year = "1992",
doi = "10.1016/0166-6851(92)90130-C",
language = "English (US)",
volume = "55",
pages = "95--104",
journal = "Molecular and Biochemical Parasitology",
issn = "0166-6851",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - The translation initiation site of recombinant Trypanosoma brucei ornithine decarboxylase varies with different promoters

AU - Kuntz, Douglas A.

AU - Phillips, Margaret A.

AU - Moore, Tracey D E

AU - Craig, Sydney P.

AU - Bass, Kathryn E.

AU - Wang, Ching C.

PY - 1992

Y1 - 1992

N2 - Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the λ phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the λ PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5′-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the λ PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the phoA promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.

AB - Expression of the Trypanosoma brucei ornithine decarboxylase (ODC) gene in Escherichia coli behind the λ phage PR promoter led to the production of a recombinant enzyme having the same subunit molecular weight as the native enzyme [4]. However, when the same gene is expressed behind the tac promoter or the phoA promoter, the ODCs produced by the transformed E. coli have subunit molecular weights approximately 2 kDa higher than that of the native enzyme. Amino terminal sequencing of the recombinant proteins indicates that the ODC synthesized under control of the λ PR promoter actually starts at the second methionine (Met23) of the open reading frame, whereas those produced in the latter two cases begin at the first methionine (Met1). Analysis of the 5′-end of T. brucei ODC mRNA supports the conclusion that translation initiates at Met23. We postulate that, for the λ PR promoter, translation initiates at Met23 instead of Met1 because of the formation of a stable secondary structure in the region of the Met1 and the presence of a good E. coli consensus translation initiation site upstream of Met23. We have constructed a new plasmid using the phoA promoter to express recombinant T. brucei ODC starting at Met23 in large quantities.

KW - mRNA structure

KW - Ornithine decarboxylase

KW - Recombinant expression

KW - Trans-splicing

KW - Trypanosoma brucei

UR - http://www.scopus.com/inward/record.url?scp=0026722635&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026722635&partnerID=8YFLogxK

U2 - 10.1016/0166-6851(92)90130-C

DO - 10.1016/0166-6851(92)90130-C

M3 - Article

C2 - 1435879

AN - SCOPUS:0026722635

VL - 55

SP - 95

EP - 104

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 1-2

ER -