Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein

Abstract

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.

The prolyl oligopeptidase family

“…Purification of the recombinant enzyme expressed in Escherichia coli is more readily achieved compared with the isolation from different tissues [15]. Prolyl oligopeptidase has been cloned from different sources, including porcine brain [16], Flavobacterium meningosepticum [17][18][19], human lymphocytes [20,21], mouse brain [22], bovine brain [23], Sarcophaga peregrina [24], Aeromonas hydrophila [25] and Pyrococcus furiosus [26,27]. The structure and localization of the mouse prolyl oligopeptidase gene has also been accomplished [28].…”

Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheat

“…Thus, significant absorbance at 410 nm by the experimental samples containing the enzyme compared to the control, which did not contain the enzyme, confirmed hydrolysis of the post-proline bond by the enzyme. The ZGPpNA substrate has been frequently used to characterize prolyl endopeptidases (Yoshimoto et al, 1978;Heins et al, 1988;Kalwant and Porter, 1991;Chevallier et al, 1992;Diefenthal et al, 1993;Harwood et al, 1997;Fulop et al, 1998;Shan et al, 2004). Use of this peptide as substrate also suggests that the specificity of the enzyme may not be dependent on which residue is present at the C-terminus of the proline as the enzyme accommodated the pNA moiety directly coupled to the carboxy terminus of the proline residue.…”

Proteolytic inventory of Thermococcus kodakaraensis

“…They were discovered in human uterus and were known as oxytocin-degrading enzymes (Walter, 1971;Koida and Walter, 1976). Prolyl olgipeptodases have been characterized from various sources including E. coli, Flavobacterium meningosepticum, Sarcophagi peregrine, Aeromonas hydrophila, P. furiosus, human lymphocytes and mouse brain (Szeltner et al, 2000;Yoshimoto et al, 1991;Diefenthal et al, 1993;Ohtsuki et al, 1997;Robinson et al, 1995;Harwood and Schreier, 2001;Vanhoff et al, 1994;Shirasawa et al, 1994). There is also found an ORF encoding prolyl olgipeptodase in the genome of T. kodakaraensis.…”