PRODUCTION OF HIGH-LEVELS OF SOLUBLE RECOMBINANT STREPTOMYCES-CLAVULIGERUS ISOPENICILLIN-N SYNTHASE IN ESCHERICHIA-COLI

Streptomyces clavuligerus isopenicillin N synthase (scIPNS) gene expre ssion under the control of T7- and trc-promoters in pET24d and pTrc99A vectors respectively in Escherichia coli was found to be affected by temperature. Although the scIPNS protein is mostly aggregated and inac tive in the inclusion bodies when made at 37 degrees C, soluble enzyme is synthesized at 25-28 degrees C. Studies conducted demonstrated tha t the promoter, as well as the E. coli strains used play critical role s in determining the level of soluble scIPNS made. It is also apparent from computational analysis that the protein structure (perhaps influ enced by hydrophobic residues at strategic positions) may also affect the solubility of the expressed scIPNS. However, after genetic manipul ation (or under appropriate conditions), overproduction of the scIPNS protein by the T7-promoter to a level of approximate to 29% of total s oluble protein in E. coli BL21(DE3) grown at 25 degrees C was achieved and the recombinant enzyme was found to retain activity. It was also observed that soluble scIPNS expressed at 25 degrees C was converted t o the insoluble form after incubation in vitro at 37 degrees C, wherea s insoluble scIPNS expressed at 37 degrees C remained aggregated regar dless of the incubation temperature in vitro. This suggested that the host's milieu affects the solubility (or folding) of the scIPNS expres sed.