Secretion by overexpression and purification of the water-soluble Streptomyces K15 DD-transpeptidase/penicillin-binding protein.
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.
Palomeque-Messia
P
P
Centre d'Ingénierie des Protéines, Université de Liège, Belgium.
Quittre
V
V
Leyh-Bouille
M
M
Nguyen-Distèche
M
M
Gershater
C J
CJ
Dacey
I K
IK
Dusart
J
J
Van Beeumen
J
J
Ghuysen
J M
JM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D015870:Gene Expression; D006602:Hexosyltransferases; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D010957:Plasmids; D012965:Sodium Chloride; D012995:Solubility; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D012965:Sodium Chloride; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase