title: A small variance in the antigenicity but not function of recombinant β-lactoglobulin purified from the culture supernatant of transformed yeast cells.
abstract: We purified recombinant bovine β-lactoglobulin (rβ-LG) from the culture supernatant of transformed yeast and investigated whether rβ-LG maintained the functional ability and antigenicity of native β-LG. Immunostaining following gel electrophoresis and reversed-phase high-performance liquid chromatography confirmed that rβ-LG was purified homogeneously. rβ-LG showed almost the same retinol-binding ability as native β-LG purified from bovine milk. However, affinities of two anti-β-LG monoclonal antibodies (mAbs) to rβ-LG were different from those to native β-LG, although three other mAbs bound these two proteins equally. Since our panel of five mAbs has been previously shown to be able to detect structural changes occurring in β-LG, this variance in antigenicity can be attributed to conformational differences between rβ-LG and native β-LG. Then, we studied which step in the production and purification procedure was responsible for altering the antigenicity of rβ-LG. Bovine milk native β-LG was added to several steps in this procedure and purified in the same manner as rβ-LG. The results suggested that incubation in the yeast culture had adverse effects on maintaining the antigenicity of this recombinant protein. We conclude from these results that even if no difference between the native and recombinant proteins can be detected by functional analysis, some subtle conformational change which can be distinguished by mAbs may be incorporated into the recombinant protein during its production and ultimately cause a different immune reaction in vivo.Abbreviations β-LG, β-lactoglobulin; rβ-LG, recombinant β-LG; PBS, phosphate-buffered saline; PBS-Tween, PBS containing 0.05% Tween 20; ELISA, enzyme-linked immunosorbent assay.
authors.affiliation: Department of Applied Biological Chemistry, The University of Tokyo, Tokyo, 113, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Katakura
authors.forename: M
authors.initials: M
authors.lastname: Totsuka
authors.forename: A
authors.initials: A
authors.lastname: Ametani
authors.forename: S
authors.initials: S
authors.lastname: Kaminogawa