Production of recombinant human type I procollagen homotrimer in the mammary gland of transgenic mice.
The large scale production of recombinant collagen for use in biomaterials requires an efficient expression system capable of processing a large (> 400 Kd) multisubunit protein requiring post-translational modifications. To investigate whether the mammary gland of transgenic animals fulfills these requirements, transgenic mice were generated containing the alpha S1-casein mammary gland-specific promoter operatively linked to 37 Kb of the human alpha 1(I) procollagen structural gene and 3' flanking region. The frequency of transgenic lines established was 12%. High levels of soluble triple helical homotrimeric [(alpha 1)3] type I procollagen were detected (up to 8 mg/ml) exclusively in the milk of six out of 9 lines of lactating transgenic mice. The transgene-derived human procollagen chains underwent efficient assembly into a triple helical structure. Although proline or lysine hydroxylation has never been described for any milk protein, procollagen was detected with these post-translational modifications. The procollagen was stable in milk; minimal degradation was observed. These results show that the mammary gland is capable of expressing a large procollagen gene construct, efficiently assembling the individual polypeptide chains into a stable triple helix, and secreting the intact molecule into the milk.
Toman
P D
PD
Cohesion Technologies, Palo Alto, CA 94303, USA. dtoman@cson.com
Pieper
F
F
Sakai
N
N
Karatzas
C
C
Platenburg
E
E
de Wit
I
I
Samuel
C
C
Dekker
A
A
Daniels
G A
GA
Berg
R A
RA
Platenburg
G J
GJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D019281:Dimerization; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000737:chemistry; D011347:Procollagen / Q000737:chemistry / Q000502:physiology*; D011392:Proline / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
D000596:Amino Acids; D011347:Procollagen; D011994:Recombinant Proteins; D011392:Proline; D008239:Lysine