High level expression of Acinetobacter calcoaceticus mutarotase in Escherichia coli is achieved by improving the translational control sequence and removal of the signal sequence

Summary The expression of enzymatically active Acinetobacter calcoaceticus encoded mutarotase in Escherichia coli is increased 10-fold upon fusion of the mro reading frame to an efficient ribosome binding sequence and deletion of the signal sequence. This was demonstrated by fusing the mro gene to the highly expressed tetR gene under control of the strong phage promoter P_L(Oehmichen et al. 1984 EMBO J. 3:539–543). Deleting the leader sequence yielded cytoplasmic expression of 5×10⁴ u of active mutarotase while constructions bearing the leader sequence expressed only 2×10³ u active enzyme. It was shown by protein and Western blot analysis that the amounts of protein expressed are nearly the same with and without signal sequence. However, under the conditions of P_Linduction the major portion of the wild type mutarotase expressed at high level remains insoluble and is not processed nor active while at low level of expression processing is complete to yield active mutarotase. The implications of these results on production of pharmaceutically interesting proteins in E. coli are discussed.