RESUMO
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.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Expressão Gênica , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Membrana Celular/enzimologia , Estabilidade Enzimática , Temperatura Alta , Dados de Sequência Molecular , Peso Molecular , Muramilpentapeptídeo Carboxipeptidase/isolamento & purificação , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Plasmídeos , Cloreto de Sódio , Solubilidade , Streptomyces/genética , Transformação BacterianaRESUMO
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.
