A SMALL VARIANCE IN THE ANTIGENICITY BUT NOT FUNCTION OF RECOMBINANT BETA-LACTOGLOBULIN PURIFIED FROM THE CULTURE SUPERNATANT OF TRANSFORMED YEAST-CELLS

We purified recombinant bovine beta-lactoglobulin (r beta-LG) from the culture supernatant of transformed yeast and investigated whether r b eta-LG maintained the functional ability and antigenicity of native be ta-LG. Immunostaining following gel electrophoresis and reversed-phase high-performance liquid chromatography confirmed that r beta-LG was p urified homogeneously. r beta-LG showed almost the same retinol-bindin g ability as native beta-LG purified from bovine milk. However, affini ties of two anti-beta-LG monoclonal antibodies (mAbs) to r beta-LG wer e different from those to native beta-LG, although three other mAbs bo und these two proteins equally. Since our panel of five mAbs has been previously shown to be able to detect structural changes occurring in beta-LG, this variance in antigenicity can be attributed to conformati onal differences between r beta-LG and native beta-LG. Then, we studie d which step in the production and purification procedure was responsi ble for altering the antigenicity of r beta-LG. Bovine milk native bet a-LG was added to several steps in this procedure and purified in the same manner as r beta-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 detec ted by functional analysis, some subtle conformational change which ca n be distinguished by mAbs may be incorporated into the recombinant pr otein during its production and ultimately cause a different immune re action in vivo.