Back to Search
Start Over
Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat
Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat
- Source :
- European Journal of Biochemistry, European Journal of Biochemistry, Wiley, 1999, 260, pp.861-868, HAL
- Publication Year :
- 1999
- Publisher :
- HAL CCSD, 1999.
-
Abstract
- The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.
- Subjects :
- Genetic Vectors
Biology
medicine.disease_cause
Biochemistry
law.invention
03 medical and health sciences
law
Escherichia coli
medicine
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Cloning, Molecular
Tyrosine
Binding site
Site-directed mutagenesis
Triticum
Plant Proteins
030304 developmental biology
0303 health sciences
Binding Sites
Expression vector
030302 biochemistry & molecular biology
Biological Transport
Lipid metabolism
Antigens, Plant
Lipid Metabolism
Molecular biology
Recombinant Proteins
Molecular Weight
Mutagenesis, Site-Directed
Recombinant DNA
Carrier Proteins
Plant lipid transfer proteins
Subjects
Details
- Language :
- English
- ISSN :
- 00142956 and 14321327
- Database :
- OpenAIRE
- Journal :
- European Journal of Biochemistry, European Journal of Biochemistry, Wiley, 1999, 260, pp.861-868, HAL
- Accession number :
- edsair.doi.dedup.....3bba7adc5815b234fc4e71614aab56ea