Open AccessCytoplasmic high‐level expression of a soluble, enzymatically active form of the Escherichia coli penicillin‐binding protein 5 and purification by dye chromatography
Author(s) -
LINDEN Mark P. G.,
MOTTL Harald,
KECK Wolfgang
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb16624.x
Subject(s) - escherichia coli , biochemistry , cytoplasm , lysis , microbiology and biotechnology , biology , cytosol , gene expression , gene , enzyme
High‐level expression of a soluble form of penicillin‐binding protein 5 (PBP5), called PBP5 s , 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 the murein polymer can be avoided by accumulation of the overexpressed protein in the cytoplasm. The signal peptide of the structural gene dac A s , coding for PBP5 s was deleted by creating a Bam HI site at the site of processing, and the truncated gene dac A sc was cloned under the control of the lambda P R promoter. Temperature induction resulted in a 200‐fold overproduction of the mature PBP5 s in the cytosol (PBP5 sc ) which is no longer harmful to the cells. PBP5 sc 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 [ 14 C]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 PBP5 sc .