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Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology.
- Source :
-
Combinatorial chemistry & high throughput screening [Comb Chem High Throughput Screen] 2001 Nov; Vol. 4 (7), pp. 545-52. - Publication Year :
- 2001
-
Abstract
- Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561-568). To identify other unknown peroxisomal enzymes, we further exploited this promising approach. Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1). Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family. However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate. The recombinant protein interacted with HsPex5p, the human PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted. Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1. These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.
- Subjects :
- Amino Acid Sequence
Animals
Cloning, Molecular
Escherichia coli metabolism
In Vitro Techniques
Molecular Sequence Data
Rabbits
Saccharomyces cerevisiae metabolism
Bacteriophage M13 enzymology
Fatty Acid Desaturases metabolism
Oxidoreductases Acting on CH-CH Group Donors
Peroxisomes enzymology
Viral Fusion Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1386-2073
- Volume :
- 4
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Combinatorial chemistry & high throughput screening
- Publication Type :
- Academic Journal
- Accession number :
- 11669066
- Full Text :
- https://doi.org/10.2174/1386207013330832