EXPRESSION OF BOVINE BETA-LACTOGLOBULIN HUMAN ERYTHROPOIETIN FUSION PROTEIN IN THE MILK OF TRANSGENIC MICE AND RABBITS

We have generated several transgenic mouse lines and rabbits expressin g efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusio n protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide e ncoding the cleavage site for bacterial IgA protease. RNA analysis per formed on one lactating transgenic mouse and one transgenic rabbit rev ealed that the fusion gene was expressed almost exlusively in the mamm ary gland, although low amounts of transgene-derived RNA were detectab le in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part ob tained upon digestion had a lower molecular mass than recombinant eryt hropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a differ ent type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon diges tion of the fusion protein with IgA proteose, biological activity comp arable to that of the recombinant erythropoietin was recovered. Transg enic males and virgin females did not show signs of enhanced erythropo iesis, but lactating females expressing the transgene displayed transi ent increases in their hematocrit values.