High-level production of the EcoRI endonuclease under the control of the pL promoter of bacterio-phage lambda

Abstract

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using λ p L promoter expression vectors. In a first step we constructed endRI :: lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the p L promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing Eco RI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the over-produced EcoRI endonuclease.