Autocatalytic processing of the TPR Thiol Protease from Porphyromonas gingivalis

Abstract

Objectives: Characterize the expression characteristics, biophysical properties and biochemical activities of the P. gingivalis Tpr protein. Methods: The tpr (PG1055) gene from P. gingivalis W83 was cloned into pGEX-6P1 and pET28a expression vectors, to encode N-terminal glutathione S-transferase (GST-Tpr) and C-terminal hexahistidine (Tpr-His6) fusion proteins, respectively. Point mutations of conserved active site residues were created (as N-terminal GST-fusions). Proteins were expressed in Escherichia coli BL21(DE3) and purified using affinity chromatography followed by size exclusion chromatography. The N-terminal GST-tag was removed during the purification process. Tpr proteolytic activities were determined using the EnzChek Gelatinase/Collagenase Assay kit (Life Technologies) with minor modifications. Results: The recombinant Tpr proteins were expressed from both vectors as a mixture of full-length (ca. 55 kDa) and N-terminally-truncated (ca. 45 kDa) forms. Chromatographic and SDS-PAGE analysis revealed that the full-length Tpr protein remained strongly associated with the N-terminal fragment (ca. 10 kDa) and truncated (ca. 45 kDa) Tpr form. The purified recombinant Tpr protein complex had efficient collagenase activities. Notably, the active-site mutants of Tpr, which completely lacked proteolytic activities, were expressed exclusively as full-length forms. This implied proteolytic self-processing occurred for the wild type Tpr protein. N-terminal (Edman) sequencing of the ca. 45 kDa Tpr band indicated autocatalytic processing had occurred in a highly specific manner. Conclusions: The initially-expressed Tpr protease cleaves itself in a specific manner yielding two protein fragments. The biological significance of this autocatalytic processing remains to be determined. Support Funding Agency/Grant Number: Research Grants Council of Hong Kong, General Research Fund (# 780713)