High-level expression of a soluble form of penicillin-binding protein 5 (PBP5), called PBP5s, and translocation across the cytoplasmic membrane results in lysis of Escherichia coli cells. The detrimental effect of increased amounts of this D,D-carboxypeptidase on the stability of murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dacAs, coding for PBP5s was deleted by creating a BamHI site at the site of processing and the truncated gene dacAsc was cloned under the control of the lambda PR promoter. Temperature induction resulted in a 200-fold overproduction of the mature PBP5s in the cytosol (PBP5sc) which is no longer harmful to the cells. PBP5sc could quantitatively be recovered in the soluble fraction after disrupting the cells. The protein retained full enzymatic activity as measured by the release of D-alanine from bisacetyl-L-Lys-D-Ala-D-Ala and formation of [14C]penicillin-protein complex at a 1:1 stoichiometry. A one-step purification procedure using the immobilized dye Procion rubine MX-B resulted in homogeneous preparations of both wild-type and mutated forms of PBP5sc.