Overexpression in Pichia pastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthora cryptogea.
A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris. Having first optimized the gene construction for secretion, we have overexpressed a modified beta-cryptogein in a secreted form. A purification scheme suited to this expression system has been developed and highly pure, biologically active protein has been obtained. For structural analysis of this recombinant beta-cryptogein, and new mutated forms thereof, optimal conditions for the crystallization of this protein have been determined and crystals that diffract to 2.2 A have been obtained.
O'Donohue
M J
MJ
Unité de Recherche de Biochimie et Structure des Protéines, INRA, Domaine de Vilvert, Jouy-en-Josas Cedex, F-78352, France.
Boissy
G
G
Huet
J C
JC
Nespoulous
C
C
Brunie
S
S
Pernollet
J C
JC
D020418:Algal Proteins; D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis
D020418:Algal Proteins; D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; C061032:cryptogein protein, Phytophthora cryptogea