Open AccessProduction in Escherichia coli and site‐directed mutagenesis of a 9‐kDa nonspecific lipid transfer protein from wheat
Author(s) -
LullienPellerin Valérie,
Devaux Carine,
Ihorai Tania,
Marion Didier,
Pahin Valérie,
Joudrier Philippe,
Gautier MarieFrançoise
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00229.x
Subject(s) - mutant , escherichia coli , biochemistry , site directed mutagenesis , biology , plant lipid transfer proteins , recombinant dna , inclusion bodies , mutagenesis , tyrosine , methionine , polyclonal antibodies , microbiology and biotechnology , amino acid , gene , genetics , antibody
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.