title: Expression of a biologically active GFP-α(S1)-casein fusion protein in Lactococcus lactis.
abstract: In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to α(S1)-casein (GFP-α(S1)Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and α(S1)-casein, a major cow's milk allergen, and the resulting vector, pNZ8148#2-GFP-α(S1)Cas, was applied to the expression of recombinant GFP-α(S1)Cas protein (rGFP-α(S1)Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-α(S1)Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from α-casein (≥70 % α(S)-casein)-immunized mice with rGFP-α(S1)Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to α-casein. Our results suggest that the expression of rGFP-α(S1)Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.
authors.affiliation: Graduate School of Agriculture, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan.
authors.forename: Suguru
authors.initials: S
authors.lastname: Shigemori
authors.forename: Shinichi
authors.initials: S
authors.lastname: Yonekura
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.forename: Maya
authors.initials: M
authors.lastname: Nakanishi
authors.forename: Hajime
authors.initials: H
authors.lastname: Otani
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimosato
mesh_terms: D000818:Animals; D002364:Caseins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006967:Hypersensitivity / Q000473:pathology; D007158:Immunologic Techniques / Q000379:methods*; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D018418:Th2 Cells / Q000276:immunology
chemical_list: D002364:Caseins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins