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 expressing 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 fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different 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 digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.
Korhonen
V P
VP
A.I. Virtanen Institute, University of Kuopio, Finland. vkorhone@messi.uku.fi
Tolvanen
M
M
Hyttinen
J M
JM
Uusi-Oukari
M
M
Sinervirta
R
R
Alhonen
L
L
Jauhiainen
M
M
Jänne
O A
OA
Jänne
J
J
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002417:Cattle; D006224:Cricetinae; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013045:Species Specificity; D014407:Tumor Cells, Cultured
D007782:Lactoglobulins; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin