Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12.

Abstract

The thioredoxin gene (trxA) from Escherichia coli K12 has been cloned on a 3-kilobase pair PvuII fragment in a derivative of pBR325 (pBHK8). Thioredoxin protein production was amplified 150-200-fold in a strain containing pBHK8 (SK3981), with the greatest increase/cell observed after cultures reached stationary phase. A simple purification procedure, involving DEAE and AcA-54 column chromatography, yielded homogeneous protein with approximately 70% yield. The high amplification of thioredoxin in these cells (i.e. 10(6) copies/cell representing 40% of total cell protein) approaches the maximum yields seen in genetically constructed cloning vehicles (Bernard, H.U., and Helinski, D.R. (1980) in Genetic Engineering (Setlow, J. K., and Hollaender, A., eds) Vol. 2, pp. 133-167, Plenum Press, New York). This tremendous overproduction of thioredoxin protein is attributed to the high plasmid copy number observed in SK3981 (1700/cell). These results suggest a role for thioredoxin in plasmid DNA replication.