Spider silks are protein-based “biopolymer” filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to “biomimic” the process of spider silk production by expressing in mammalian cells the dragline silk genes ( ADF-3 / MaSpII and MaSpI ) of two spider species. We produced soluble recombinant (rc)–dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc–spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc–silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat ␤-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.organophosphorus nerve agent ͉ recombinant protein expression ͉ transgenic production H uman plasma butyrylcholinesterase (huBChE) (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of Ϸ340 kDa that is stable in plasma with a half-life of Ϸ12 days (1, 2). Although the physiological function of huBChE is unclear, the enzyme prevents intoxication of animals exposed to organophosphorus (OP) compounds (3, 4). The huBChE enzyme also hydrolyzes many ester-containing drugs, such as cocaine and succinylcholine (5). The toxicity of OP agents is due to irreversible inhibition of acetylcholinesterase and the subsequent continuous stimulation of neurons by acetylcholine (6). Administration of exogenous huBChE, which irreversibly binds OP agents to prevent inactivation of acetylcholinesterase and continuous cholinergic stimulation, is a potential strategy for preventing toxicity from OP agents (4). Although huBChE has been obtained from human plasma by a large scale purification technique, this procedure is severely limited by the volume of human plasma needed (7). It is unlikely that a sufficient amount of enzyme could be purified commercially by this technique. Because of the 1:1 stoichiometry required for protection against exposure to OP agents (8), large quantities of huBChE are needed for effective prophylaxis and treatment of exposure. Compared with other potential enzymatic bioscavengers of OP agents, huBChE has a broad spectrum of activity, a relatively long half-life, and limited, if any, physiological side effects (9). Producing recombinant BChE (rBChE) is an alternative to purification of the enzyme from human plasma. Recombinant huBChE has been expressed in Escherichia coli (10), albeit in a nonfunctional form; mammalian 293T (11); and CHO (12) cells. However, these expression systems cannot economically produce sufficient quantities of rBChE with a residence time similar to native huBChE that would allow development of the enzyme as an agent for prophylaxis against OP poisoning.The production of recombinant proteins by the mammary g...

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

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