PHYSICOCHEMICAL AND BIOCHEMICAL-CHARACTERIZATION OF HUMAN ALPHA(1)-MICROGLOBULIN EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS

DNA encoding the signal peptide and the alpha(1)-microglobulin part of the human alpha(1)-microglobulin-bikunin gene was expressed in baculo virus-infected insect cells. Recombinant alpha(1)-microglobulin was se creted and could be purified from the medium with a yield of 20-30 mg/ L. Biochemical and physicochemical characterization showed that the re combinant protein was very similar to alpha(1)-microglobulin isolated from human urine and plasma, except that the recombinant protein had s maller N-linked oligosaccharides, lacked the O-Linked oligosaccharide, and was devoid of sialic acid. Recombinant alpha(1)-microglobulin mig rated upon SDS-PAGE as two bands, 27 and 29 kDa, representing alpha(1) -microglobulin with one and two N-linked carbohydrates, respectively. An overall structural similarity was indicated as antibodies raised ag ainst human urinary alpha(1)-microglobulin were found to recognize rec ombinant, plasma, and urinary alpha(1)-microglobulin in a similar mann er. CD studies suggested an almost identical secondary structure for r ecombinant and urinary alpha(1)-microglobulin but a slightly different structure for plasma alpha(1)-microglobulin. The absorbance spectrum as well as visual examination demonstrated that recombinant, urinary, and plasma alpha(1)-microglobulin carried a yellow-brown chromophore, but that plasma alpha(1)-microglobulin was slightly less intensely col ored. Although it is still a puzzle why the immunosuppressive plasma p rotein alpha(1)-microglobulin and the protease inhibitor bikunin, whic h have no known function in common, are cotranslated from the same mRN A, it can be concluded that bikunin is not necessary for an adequate t ranslation, folding, and secretion of alpha(1)-microglobulin. Furtherm ore, since recombinant alpha(1)-microglobulin was produced in large am ounts and found to be very similar to plasma and urinary alpha(1)-micr oglobulin, it may prove to be useful in structural and functional stud ies of the protein. (C) 1997 Academic Press.