Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat.
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.
Lullien-Pellerin
V
V
Unité de Biochimie et Biologie Moléculaire des Céréales, INRA, Montpellier, France. lullien@ensam.inra.fr
Devaux
C
C
Ihorai
T
T
Marion
D
D
Pahin
V
V
Joudrier
P
P
Gautier
M F
MF
D052179:Antigens, Plant; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D005822:Genetic Vectors; D050356:Lipid Metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014908:Triticum / Q000235:genetics*
D052179:Antigens, Plant; D002352:Carrier Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C511920:lipid transfer proteins, plant