Homologous expression of Pseudomonas aeruginosa phosphorylcholine phosphatase

BEASSONI, PR; MASSIMELLI, MJ; FORRELLAD, MA; GARRIDO, MN; LISA, AT; DOMENECH, CE

Tafí del Valle, Tucumán

Congreso; XXI Reunión anual de la Sociedad de Biología de Tucumán (SBT); 2004

Sociedad de Biología de Tucuman

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced when the bacteria are grown in the presence of choline or its derivates. In previous reports, we found that pchP heterologous expression resulted in a totally functional protein but with some kinetics differences respect the native enzyme. Therefore, the present study describes an expressing–cloning strategy employed to achieve the expression of PChP in an homologous host. This was carried out in P. aeruginosa PAO1-LAC transformed with the vector pUCP‑Nde-pchP (rPChP-Pa). rPChP-Pa exhibited a molecular mass of approximately 40 kDa, as expected for the size of the gene and catalyzes the hydrolysis of phosphorylcholine (PC), phosphoryl- ethanolamine and p-NPP. Like the purified native PChP, rPChP-Pa shown a high and low affinity sites for PC and its activity was inhibited by high substrate concentration. rPChP-Pa was much more similar to native PChP that recombinant PChP expressed in E. coli (rPChP-Ec). The reason for this differences may be the presence of signal peptide in rPChP-Ec which apparently E. coli is not capable of process as can be seen in western blot by the slight higher molecular mass observed in rPChP-Ec respect to the native PChP or rPCh-Pa enzymes.