One step purification and characterization of the pyrrolidone carboxyl peptidase of Streptococcus pyogenes over-expressed in Escherichia coli.
Pyrrolidone carboxyl peptidase (EC 3.4.11.8) (Pcp), an enzyme which selectively removes pyrrolidone carboxylic acid (PCA) from some PCA-peptides and -proteins, was demonstrated in bacteria and in plant, animal and human tissues. In this paper we describe the purification to homogeneity of the enzyme of Streptococcus pyogenes, over-expressed in Escherichia coli. This was achieved, for the first time in one step, by hydrophobic interaction chromatography. Analysis under non-denaturing conditions revealed a molecular mass of 85 kDa and in the presence of sodium dodecyl sulfate gave a molecular mass of 23.5 kDa. Investigations on enzymatic properties showed that the Pcp over-expressed in E. coli disclosed properties similar to those found for the enzyme extracted from S. pyogenes or for some other Pcps studied previously. Thus the over-expressed enzyme should serve as a suitable source for N-terminal unblocking prior to some PCA protein sequencing.
Laboratoire de Génétique Moléculaire des Microorganismes, Institut National des Sciences Appliquées, Villeurbanne, France.
A
A
Awadé
T
T
Gonzalès
P
P
Cleuziat
J
J
Robert-Baudouy
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D010957:Plasmids; D011751:Pyroglutamyl-Peptidase I / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology*
D011751:Pyroglutamyl-Peptidase I