Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei sequence

Expression and molecular analysis

Roger Pellé, Vern L. Schramm, David W. Parkin

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size- polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in blood-stream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

Original languageEnglish (US)
Pages (from-to)2118-2126
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number4
DOIs
StatePublished - Jan 23 1998

Fingerprint

Trypanosoma brucei brucei
Cloning
Molecular Cloning
Genes
Amino Acids
Open Reading Frames
Amino Acid Sequence
Trypanosoma vivax
Crithidia fasciculata
Trypanosoma brucei gambiense
Trypanosoma brucei rhodesiense
Trypanosoma congolense
Genome
Trypanosoma
Salvaging
Trypanosoma cruzi
Haploidy
Chromosomes
Southern Blotting
inosine-adenosine-guanosine-preferring nucleoside hydrolase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei sequence : Expression and molecular analysis. / Pellé, Roger; Schramm, Vern L.; Parkin, David W.

In: Journal of Biological Chemistry, Vol. 273, No. 4, 23.01.1998, p. 2118-2126.

Research output: Contribution to journalArticle

@article{c790aa8b73424d85ab19d57591ac8098,
title = "Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei sequence: Expression and molecular analysis",
abstract = "N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size- polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in blood-stream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97{\%} homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.",
author = "Roger Pell{\'e} and Schramm, {Vern L.} and Parkin, {David W.}",
year = "1998",
month = "1",
day = "23",
doi = "10.1074/jbc.273.4.2118",
language = "English (US)",
volume = "273",
pages = "2118--2126",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "4",

}

TY - JOUR

T1 - Molecular cloning and expression of a purine-specific N-ribohydrolase from Trypanosoma brucei brucei sequence

T2 - Expression and molecular analysis

AU - Pellé, Roger

AU - Schramm, Vern L.

AU - Parkin, David W.

PY - 1998/1/23

Y1 - 1998/1/23

N2 - N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size- polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in blood-stream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

AB - N-Ribohydrolases, including the inosine-adenosine-guanosine-preferring (IAG) nucleoside hydrolase, have been proposed to be involved in the nucleoside salvage pathway of protozoan parasites and may constitute rational therapeutic targets for the treatment of these diseases. Reported is the complete sequence of the Trypanosoma brucei brucei iagnh gene, which encodes IAG-nucleoside hydrolase. The 1.4-kilobase iagnh cDNA contains an open reading frame of 981 base pairs, corresponding to 327 amino acids. The iagnh gene is present as one copy/haploid genome and is located on the size- polymorphic pair of chromosome III or IV in the genome of T. b. brucei. In Southern blot analysis, the iagnh probe hybridized strongly with Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Trypanosoma evansi, Trypanosoma congolense, and Trypanosoma vivax and, to a lesser extent, with Trypanosoma cruzi genomic DNA. The iagnh gene is expressed in blood-stream forms and procyclic (insect) life-cycle stages of T. b. brucei. There are no close amino acid homologues of IAG-nucleoside hydrolase outside bacterial, yeast, or parasitic organisms. Low amino acid sequence similarity is seen with the inosine-uridine-preferring nucleoside hydrolase isozyme from Crithidia fasciculata. The T. b. brucei iagnh open reading frame was cloned into Escherichia coli BL21(DE3), and a soluble recombinant IAG-nucleoside hydrolase was expressed and purified to >97% homogeneity. The molecular weights of the recombinant IAG-nucleoside hydrolase, based on the amino acid sequence and observed mass, were 35,735 and 35,737, respectively. The kinetic parameters of the recombinant IAG-nucleoside hydrolase are experimentally identical to the native IAG-nucleoside hydrolase.

UR - http://www.scopus.com/inward/record.url?scp=0031890380&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031890380&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.4.2118

DO - 10.1074/jbc.273.4.2118

M3 - Article

VL - 273

SP - 2118

EP - 2126

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 4

ER -