Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography

Abstract

The absorption of thymidylate synthetase from Escherichia coli B to aminoalkyl-Sepharose with the increasing length of carbon chain (2-6 C) was investigated. A correlation was found between the chain length and absorption effectiveness, increasing from the 2 to the 6 C chain. A hydrophobic chromatography of the enzyme on aminobutyl-Sepharose gave .apprx. 20-fold purification. A new affinity chromatography carrier was synthesized containing tetrahydromethotrexate linked to aminoethyl-Sepharose via its carboxylic groups. The carrier absorbed the enzyme from the crude preparation only in the presence of 2 .times. 10-5 M dUMP. The specifically absorbed thymidylate synthetase was eluted with sacharose-containing buffers in which dUMP was omitted. The purification procedure was applied to a crude thymidylate synthetase preparation from resting E. coli, calf thymus, Sarcoma 180 [mouse] and Gardner lymphosarcoma. The purified enzyme from all mentioned sources showed 1 protein band on disc electrophoresis corresponding to enzymatic activity. The formation of a reversible noncovalent complex enzyme-tetrahydromethotrexate-dUMP on the affinity column is considered.