The purpose of this research is to prepare an immobilized protein synthesis system based on in vitro translation of messenger RNA. This research is, in a way, an extension of the investigators laboratory's interest in solid phase biochemistry and biomolecule immobilization. The investigators will formulate their initial attempts to prepare an immobilized protein synthesis system based on two observations. The first is the development by Dr. Spirin of the Soviet Union of a continuous flow immobilized protein synthesis system formed when a rabbit reticulocyte or wheat germ protein synthesis system is separated from a feed stock of amino acids and nucleoside triphyosphates by a semipermeable membrane which permits the exit of small newly synthesized proteins but does not permit the exit of the protein synthesis complex. The other investigation on which this system will be based is the two-phased aqueous partitioning systems of Albertsson in which the investigators will employ, for example, polyethylene glycol and dextran to partition the protein synthesis system into one phase and allow the small molecules, such as amino acids and ATP, to enter from the other phase. Eventually, the investigators will implant a two-phase aqueous protein immobilization system within an asymmetric hollow fiber such that the outside large pore side of the hollow fiber will contain the phase which does not hold the in vitro translation system, whereas the lumen of the hollow fiber will be too small for the components of the in vitro translation system to penetrate. The small molecules needed for protein synthesis, such as ATP and amino acids, will be infused through the lumen of the system. The product protein will be removed by affinity partitioning using a polymeric ligand, such as polyethylene glycol-ligand, contained in the phase surrounding the outside of the hollow fiber. This will be used to remove the protein product as soon as it is translated. In order to prevent the removal of polysomes, it may be necessary to immobilize the messenger RNA onto the internal surface of the hollow fiber. Eventually, a second apparatus will be created through which the external phase passes and the protein being synthesized will be recovered from the ligand and the polymeric polyethylene glycol-ligand phase returned to the external side of the hollow fiber. This represents an extremely novel approach to cell free protein synthesis and has a very high potential to permit the production of highly purified protein products for a variety of therapeutic and non-therapeutic applications. Although such applications are not within the scope of this proposal, the investigator intends to utilize both simple model messenger RNAs in the development of the cell free protein synthesis system and to apply it later to the synthesis of several high value therapeutic proteins such as insulin and leukokines.
| Marszal, E; Scouten, W H (1996) Dihydrofolate reductase synthesis in the presence of immobilized methotrexate. An approach to a continuous cell-free protein synthesis system. J Mol Recognit 9:543-8 |
