ABSTRACT
Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.
Subject(s)
Bacterial Proteins , Carrier Proteins/genetics , Gene Expression , Hexosyltransferases , Muramoylpentapeptide Carboxypeptidase/genetics , Peptidyl Transferases , Streptomyces/enzymology , Amino Acid Sequence , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Membrane/enzymology , Enzyme Stability , Hot Temperature , Molecular Sequence Data , Molecular Weight , Muramoylpentapeptide Carboxypeptidase/isolation & purification , Muramoylpentapeptide Carboxypeptidase/metabolism , Penicillin-Binding Proteins , Penicillins/metabolism , Plasmids , Sodium Chloride , Solubility , Streptomyces/genetics , Transformation, BacterialABSTRACT
A method of using plate exchange equipment for the culture of anchorage dependent cells is described. No special orientation of the plates is required and cells may be readily recovered for subsequent use in other culture vessels. The method offers certain advantages over existing systems.
