EXPRESSION OF RAT ALPHA(1)-MICROGLOBULIN-BIKUNIN IN BACULOVIRUS-TRANSFORMED INSECT CELLS

cDNA encoding rat a(1)-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting a(1)-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted a(1)-microglobulin with maximal concentrations of 15 mg/liter 5 days after infection. The secreted proteins migrated upon SDS-PAGE as two major protein bands, 40 and 26 kDa, corresponding to a(1)-microglobulin-bikunin and free a(1)-microglobulin. The results suggested that the cells secreted mostly a(1)-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free a(1)-microglobulin. Both forms were isolated by monoclonal anti-a(1)-microglobulin affinity chromatography, and a(1)-microglobulin-bikunin separated from free a(1)-microglobulin by gel chromatography. The yields of purified a(1)-microglobulin-bikunin and free a(1)-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell a(1)-microglobulin displayed a size, shape, and charge heterogeneity similar to a(1)-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary a(1)-microglobulin from several different species bound to rat urinary a(1)-microglobulin and insect cell secreted a(1)-microglobulin-bikunin and free a(1)-microglobulin with approximately the same strength, indicating that the three proteins are folded in similar ways, The results of glycosidase treatments and lectin blotting indicate the absence of neuraminic acid but the presence of one N-linked oligosaccharide and an unspecified number of O-linked oligosaccharides in a(1)-microglobulin-bikunin and free a(1)-microglobulin. (C) 1995 Academic Press, Inc.