333 HIGH LEVEL EXPRESSION OF BIOACTIVE RECOMBINANT HUMAN GROWTH HORMONE IN THE MILK OF A CLONED TRANSGENIC COW
Authors: Salamone D.F. , Barañao J.L. , Santos C.B. , Bussman L. , Artuzo J. , Carboneto C. , Paupuchado M. , Criscuolo M. 
Published: December 2004

Transgenic farm animals have been proposed as an alternative to current bioreactors by expression in E. coli but no comparisons have been made yet for the the production of the same protein by both methods. Recombinant growth hormone (hGH) was produced by fermentation in E. coli by a procedure routinely used for the commercial production of the hormone. Fetal fibroblasts were obtained from a Jersey 75-day fetus and transfected with separate plasmids containing constructs comprising the human growth hormone gene (650 bp cDNA) under the control of a bovine β-casein promoter and a neomycin resistance gene. Control was a nontransgenic parental cell line; all other groups were selected for 14 days with 800 μg/mL geneticin, and three cell lines (L0–L2) were obtained and were used as donors. NT was performed as described by Salamone et al. 2004 Reprod. Fert. Dev. 16, 158. Two blastocysts were transferred nonsurgically per recipient cow. The blastocyst numbers per total numbers of NT by treatment were: control, 33/197; L0, 28/130; L1, 34/137; and L2, 71/470. Chi-square analysis showed no significant differences among groups. Calves born were: n = 1, 4, 0, and 5, from control, L0, L1, and L2 groups, respectively. The total rate of development to term of transferred doublets was 12%. After testing by PCR and Southern Blot, only one calf from line L0 showed a complete sequence for the coding region of the hGH whereas partial deletions were observed in the other animals. Artificial lactation was induced at 11 months and milking was started on Day 24 after the start of the hormonal treatment regime. Bioactivity of hGH in the milk, measured by a Nb2 cell proliferation assay, started to increase from values around 2 g/L at the onset of milking and rose steadily, reaching values of 5.0 g/L. A highly significant correlation was observed between the bioactivity and the immunoactivity determined with a specific antibody. Analysis of whey milk showed a major band corresponding to rhGH after staining with Commassie blue. This band represents about 10% of the total protein content. In comparison, in the cell extract from E. coli, rhGH represented less than 5%. The peptidic map of rhGH from milk was identical to that of the hormone produced by bacterial fermentation. At the present production rates, which are expected to be increased after the cow reaches a natural lactation, the annual amounts of hGH produced from this single cow would be about 4,400 g, which represents a 445% increase over our yield in conventional bacterial fermentation (500 L fermentor). Only about 15 animals would be required to cover the world needs for the treatment of children suffering from dwarfism. Here we show the production of recombinant human growth hormone in the milk of a cloned transgenic cow in levels of up to 5 g/L. The hormone has identical bioactivity to that currently produced by expression in E. coli. These results demonstrate that transgenic cattle can be used as an alternative for the production of this hormone.
The authors are grateful to Biosidus for financial support and technical assistance.