title: Expression and thrombin cleavage of Poa p IX recombinant allergens fused to glutathione S-transferase.
abstract: The high-level expression and purification of Poa p IX recombinant grass pollen allergens were examined utilizing a modified pGEX plasmid, designated as pGEX 2T-1. This vector permits frame-1 ligation of lambda gt11 cDNA inserts and cleavage of the recombinant allergenic protein from the fusion partner glutathione S-transferase. The expression of the fusion proteins in water-soluble form varied among the transformants of the same bacterial strain and also between different host strains. Purification of the fusion proteins by affinity chromatography employing glutathione agarose gel revealed that proteases in the bacterial lysate bound to the gel and were co-eluted with the fusion proteins. These proteases, which specifically degraded the recombinant proteins to varying degrees, were inhibited by both of the inhibitors, phenylmethylsulfonyl fluoride and aprotinin. Cleavage by thrombin of the fusion proteins indicated that the structure of the individual protein affected the thrombin accessibility to the cleavage site. Increased concentration of thrombin partly compensated this effect, but resulted in a broader specificity of the enzyme. By contrast, cleavage of the fusion protein when it was still attached to the glutathione gel was convenient and led to purification of the product devoid of proteolytic activity. Since almost all the recombinant allergens have been cloned in lambda gt11 vector, the pGEX 2T-1 vector reported herein will facilitate the synthesis, purification of the corresponding allergenic proteins or their peptides in soluble and biologically active forms.
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Olsen
authors.forename: S S
authors.initials: SS
authors.lastname: Mohapatra
mesh_terms: D000485:Allergens / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011058:Pollen / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
chemical_list: D000485:Allergens; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D013917:Thrombin