Expression of rat alpha 1-microglobulin-bikunin in baculovirus-transformed insect cells.
cDNA encoding rat alpha 1-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting alpha 1-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted alpha 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 alpha 1-microglobulin-bikunin and free alpha 1-microglobulin. The results suggested that the cells secreted mostly alpha 1-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free alpha 1-microglobulin. Both forms were isolated by monoclonal anti-alpha 1-microglobulin affinity chromatography, and alpha 1-microglobulin-bikunin separated from free alpha 1-microglobulin by gel chromatography. The yields of purified alpha 1-microglobulin-bikunin and free alpha 1-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell alpha 1-microglobulin displayed a size, shape, and charge heterogeneity similar to alpha 1-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary alpha 1-microglobulin from several different species bound to rat urinary alpha 1-microglobulin and insect cell secreted alpha 1-microglobulin-bikunin and free alpha 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 alpha 1-microglobulin-bikunin and free alpha 1-microglobulin.
Bratt
T
T
Department of Cell and Molecular Biology, Lund University, Sweden.
Akerström
B
B
D000510:Alpha-Globulins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007526:Isoelectric Point; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D014170:Transformation, Genetic; D014360:Trypsin Inhibitor, Kunitz Soybean
D000510:Alpha-Globulins; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011480:Protease Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; C016001:alpha-1-microglobulin; D014360:Trypsin Inhibitor, Kunitz Soybean