Author: Wester L. Johansson M.U. Akerstrom B.
Publisher: Academic Press
ISSN: 1046-5928
Source: Protein Expression and Purification, Vol.11, Iss.1, 1997-10, pp. : 95-103
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Abstract
DNA encoding the signal peptide and the alpha1-microglobulin part of the human alpha1-microglobulin-bikunin gene was expressed in baculovirus-infected insect cells. Recombinant alpha1-microglobulin was secreted and could be purified from the medium with a yield of 20-30 mg/L. Biochemical and physicochemical characterization showed that the recombinant protein was very similar to alpha1-microglobulin isolated from human urine and plasma, except that the recombinant protein had smaller N-linked oligosaccharides, lacked the O-linked oligosaccharide, and was devoid of sialic acid. Recombinant alpha1-microglobulin migrated upon SDS-PAGE as two bands, 27 and 29 kDa, representing alpha1-microglobulin with one and two N-linked carbohydrates, respectively. An overall structural similarity was indicated as antibodies raised against human urinary alpha1-microglobulin were found to recognize recombinant, plasma, and urinary alpha1-microglobulin in a similar manner. CD studies suggested an almost identical secondary structure for recombinant and urinary alpha1-microglobulin but a slightly different structure for plasma alpha1-microglobulin. The absorbance spectrum as well as visual examination demonstrated that recombinant, urinary, and plasma alpha1-microglobulin carried a yellow-brown chromophore, but that plasma alpha1-microglobulin was slightly less intensely colored. Although it is still a puzzle why the immunosuppressive plasma protein alpha1-microglobulin and the protease inhibitor bikunin, which have no known function in common, are cotranslated from the same mRNA, it can be concluded that bikunin is not necessary for an adequate translation, folding, and secretion of alpha1-microglobulin. Furthermore, since recombinant alpha1-microglobulin was produced in large amounts and found to be very similar to plasma and urinary alpha1-microglobulin, it may prove to be useful in structural and functional studies of the protein.
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