Dihydrofolate reductase synthesis in the presence of immobilized methotrexate. An approach to a continuous cell-free protein synthesis system

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Abstract
Dihydrofolate reductase was synthesized in a batch system in the presence of the affinity ligand methotrexate, bound to various matrices. Two types of gel were used: commercial methotrexate–agarose with pores inaccessible for translation machinery and methotrexate–POROS with pores easily accessible for translation reaction mixture components. The transcription/translation reaction was not inhibited by either the immobilized methotrexate or the matrix. The enzyme was synthesized with a high yield and could simultaneously be removed from the reaction mixture by the affinity matrix during the synthesis. With methotrexate–POROS present the reaction probably proceeded mainly in the pores of the gel. Kinetic limitations to the reaction in the presence of the gel were not observed. Active dihydrofolate reductase was eluted from methotrexate–POROS. The activity recovered was higher than dihydrofolate reductase activity synthesized in free solution system. The influence of the presence of immobilized methotrexate on dihydrofolate reductase synthesis will be further studied in a novel type of a continuous protein synthesis system.