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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

Authors :
Didier Marion
Carine Devaux
Tania Ihorai
Philippe Joudrier
Marie-Françoise Gautier
Valérie Pahin
Valerie Lullien-Pellerin
Unité de biochimie et biologie moléculaire des céréales
Institut National de la Recherche Agronomique (INRA)
UR 0724 Unité de recherche Biochimie et technologie des protéines
Institut National de la Recherche Agronomique (INRA)-Transformation des Produits Végétaux (T.P.V.)-Unité de recherche Biochimie et technologie des protéines (NANT LBTP)
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.

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