Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1β synthesis in Escherichia coli

Abstract

A new dual-replicon recombinant plasmid, pPR53- tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121–126]. In contrast to its progenitor, pPR53- tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and alow Cop at28°C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the p L promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53- tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42°C. Thus, pPR53- tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-lβ (hIL-1β) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1β(re-hIL-1β) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the watersoluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1β. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3–5 mg ofre-hIL-lβ/g of wet cells. The re-hIL-1β specific activity was about 2 × 10 8 units/mg, coinciding with that of the authentic hIL-1β.