Taking the p... therapeutic proteins from urine
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US researchers have developed the bladder of transgenic mice as a novel bioreactor that could prove more cost-effective than milk-based and blood-based systems for producing foreign proteins [Kerr, D.E. et al. (1998) Nat. Biotechnol. 16, 75–79]. The method should be easy to scale up to large animals such as cattle for commercial production. Bob Wall and colleagues in the US Department of Agriculture (Beltsville, MD, USA) and New York University Medical School (New York, NY, USA) used part of the mouse gene for uroplakin II (UPII), a urothelial membrane protein, to direct the production of human growth hormone (hGH) in the urothelium of transgenic mice. hGH was chosen for the experiment because ectopic (‘leaky’) expression is easy to detect. ‘We know the hormone was active in the transgenic mice, because some were very large, and all the females were sterile’, says Wall. Production of pharmaceuticals by ‘biopharming’ is cheaper and easier to scale up than cell-culture based systems, and several products derived from the milk of transgenic farm animals are now under clinical trial. A major advantage of the bladder as a bioreactor, say the authors, is the ability to harvest the product throughout the life of the animal – instead of just during lactation – and without regard for the transgenic animal’s sex or reproductive status. To generate the transgenic mice, the UPII–hGH transgene (produced by ligating the 3.6 kb 5′ flanking region of the UPII mouse gene to the 5′ end of the hGH structural gene) was microinjected into fertilized eggs, which were then transferred to pseudopregnant recipients. This procedure resulted in the birth of five (sterile) female and four male founder transgenic animals. hGH was detected in the urine of three of the males, and these were bred to establish lines of mice for further study. Northern blot analysis showed ‘substantial’ expression of hGH in the bladder – two orders of magnitude higher than in brain and kidneys; and immunofluorescent staining with an antibody to hGH revealed its expression in urothelium (Fig. 1). Radioimmunoassay showed hGH concentrations of up to 500 ng ml 21 in the urine of UPII–hGH transgenic mice (and none in control mice). Concentrations were relatively stable in individual animals from the first sampling at age 6 weeks to the last at 8 months. The relative levels of expression of the founder animals were generally preserved in their offspring. Mouse urine contains about 10- 50 times as much total protein as bovine urine, mostly as so-called major urinary proteins produced by the liver. Thus, hGH represents only about 0.02% of urinary proteins in transgenic mice but could probably account for 0.1–1.0% of urinary protein in transgenic farm animals. This compares with a figure of 1–10% of total milk protein for pharmaceutical proteins produced by mammary-specific transgenes However, it is unlikely that the bladder could ever come close to producing as much protein as the mammmary gland, even if expression in urine could be enhanced. “Mammary glands really crank out a lot of protein – in the g l 21 range – whereas the bladder is making protein in the mg l 21 range” says Wall. But purification of specific proteins would be simpler from urine than from milk. At least one step essential – fat separation – would not be required for purification from urine. ‘The cost-effectiveness of purification of protein from urine will probably turn out to be the most significant benefit of a bladder bioreactor’, adds Wall. Wall’s team have no plans to produce other therapeutic proteins from urine. Their method is not being patented and is free to be used by others. Uroplakin genes are highly conserved in other mammalian species, including humans, cattle and sheep. Biologicals purified from urine are already used in clinical medicine – pregnant mares’ urine, for instance, is a major source of natural oestrogen for postmenopausal hormone replacement. In Canada and the USA, urine for this purpose is collected from 75 000 mares, each producing about 500 l of urine a year. Wall calculates that a herd of only 200 transgenic cattle could provide enough factor VIII to supply the whole world, assuming that each animal produced 20 l per day of urine containing 0.5 mg l 21 of the protein, and taking into account an estimated 50% loss of the product during purification. ‘And harvesting starts earlier with urine-based production’, adds Wall. ‘It would take about 3 years from embryo microinjection to generate a herd producing a transgene in urine, compared with 7 years for a milk-borne transgene.’ Dorothy Bonn 230 N e w s MOLECULAR MEDICINE TODAY, JUNE 1998 Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00 Figure 1. Bladder from a transgenic mouse expressing growth hormone in the urothelial layer, stained with a fluorescently labelled antibody to human growth hormone. E denotes the epithelial layer; L denotes the lumen. Reproduced from Nat. Biotechnol. 16, 75–79. Image kindly provided by Dr Robert J. Wall, US Dept of Agriculture, Beltsville, MD 20705, USA. Taking the p… therapeutic proteins from urine
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Transcript of Taking the p... therapeutic proteins from urine
US researchers have developed the bladder oftransgenic mice as a novel bioreactor that couldprove more cost-effective than milk-based andblood-based systems for producing foreignproteins [Kerr, D.E. et al. (1998) Nat.Biotechnol. 16, 75–79]. The method should beeasy to scale up to large animals such as cattlefor commercial production.
Bob Wall and colleagues in the USDepartment of Agriculture (Beltsville, MD,USA) and New York University Medical School(New York, NY, USA) used part of the mousegene for uroplakin II (UPII), a urothelialmembrane protein, to direct the production ofhuman growth hormone (hGH) in theurothelium of transgenic mice. hGH was chosenfor the experiment because ectopic (‘leaky’)expression is easy to detect. ‘We know thehormone was active in the transgenic mice,because some were very large, and all thefemales were sterile’, says Wall.
Production of pharmaceuticals by‘biopharming’ is cheaper and easier to scale upthan cell-culture based systems, and severalproducts derived from the milk of transgenicfarm animals are now under clinical trial. A
major advantage of the bladder as a bioreactor,say the authors, is the ability to harvest theproduct throughout the life of the animal –instead of just during lactation – and withoutregard for the transgenic animal’s sex orreproductive status.
To generate the transgenic mice, theUPII–hGH transgene (produced by ligating the3.6 kb 5′ flanking region of the UPII mousegene to the 5′ end of the hGH structural gene)was microinjected into fertilized eggs, whichwere then transferred to pseudopregnantrecipients. This procedure resulted in the birthof five (sterile) female and four male foundertransgenic animals. hGH was detected in theurine of three of the males, and these were bred to establish lines of mice for further study.
Northern blot analysis showed ‘substantial’expression of hGH in the bladder – two orders of magnitude higher than in brain andkidneys; and immunofluorescent staining withan antibody to hGH revealed its expression inurothelium (Fig. 1). Radioimmunoassayshowed hGH concentrations of up to 500 ng
ml21 in the urine ofUPII–hGHtransgenic mice (and none in control mice).Concentrations wererelatively stable in individual animals from the first sampling at age 6 weeks to thelast at 8 months. The relative levelsof expression of thefounder animalswere generallypreserved in theiroffspring.
Mouse urinecontains about 10-50 times as muchtotal protein asbovine urine, mostlyas so-called major
urinary proteins produced by the liver. Thus,hGH represents only about 0.02% of urinaryproteins in transgenic mice but could probablyaccount for 0.1–1.0% of urinary protein intransgenic farm animals. This compares with afigure of 1–10% of total milk protein forpharmaceutical proteins produced bymammary-specific transgenes However, it isunlikely that the bladder could ever come closeto producing as much protein as the mammmarygland, even if expression in urine could beenhanced. “Mammary glands really crank out alot of protein – in the g l21 range – whereas thebladder is making protein in the mg l21 range”says Wall. But purification of specific proteinswould be simpler from urine than from milk. Atleast one step essential – fat separation – wouldnot be required for purification from urine. ‘Thecost-effectiveness of purification of proteinfrom urine will probably turn out to be the mostsignificant benefit of a bladder bioreactor’,adds Wall.
Wall’s team have no plans to produce othertherapeutic proteins from urine. Their method isnot being patented and is free to be used byothers. Uroplakin genes are highly conserved inother mammalian species, including humans,cattle and sheep.
Biologicals purified from urine are already used in clinical medicine – pregnantmares’ urine, for instance, is a major source of natural oestrogen for postmenopausalhormone replacement. In Canada and the USA,urine for this purpose is collected from 75 000mares, each producing about 500 l of urine ayear. Wall calculates that a herd of only 200transgenic cattle could provide enoughfactor VIII to supply the whole world,assuming that each animal produced 20 l perday of urine containing 0.5 mg l21 of theprotein, and taking into account an estimated 50% loss of the product duringpurification. ‘And harvesting starts earlier withurine-based production’, adds Wall. ‘It wouldtake about 3 years from embryo microinjectionto generate a herd producing a transgene inurine, compared with 7 years for a milk-bornetransgene.’
Dorothy Bonn
230
N e w s MOLECULAR MEDICINE TODAY, JUNE 1998
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00
Figure 1. Bladder from a transgenic mouse expressing growth hormone inthe urothelial layer, stained with a fluorescently labelled antibody to humangrowth hormone. E denotes the epithelial layer; L denotes the lumen.Reproduced from Nat. Biotechnol. 16, 75–79. Image kindly provided by Dr Robert J. Wall, US Dept of Agriculture, Beltsville, MD 20705, USA.
Taking the p… therapeutic proteins from urine
