Lipid transfer proteins from fruit: cloning, expression and quantification.
BACKGROUND
Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs.


METHODS
cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3.


RESULTS
The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3.


CONCLUSIONS
rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.
Zuidmeer
Laurian
L
Department of Immunopathology, Sanquin, Amsterdam, The Netherlands.
van Leeuwen
W Astrid
WA
Budde
Ilona Kleine
IK
Cornelissen
Jessica
J
Bulder
Ingrid
I
Rafalska
Ilona
I
Besolí
Noèlia Telléz
NT
Akkerdaas
Jaap H
JH
Asero
Riccardo
R
Fernandez Rivas
Montserrat
M
Rivas
Montserrat Fernandez
MF
Gonzalez Mancebo
Eloina
E
Mancebo
Eloina Gonzalez
EG
van Ree
Ronald
R
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D019976:Cloning, Organism; D005638:Fruit / Q000276:immunology*; D027845:Malus / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins; D014176:Protein Biosynthesis; D027861:Prunus / Q000276:immunology*; D011817:Rabbits
D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; C463180:Mal d 3 allergen; D010940:Plant Proteins; C463181:Pru p 3 allergen; C511920:lipid transfer proteins, plant