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Conference Paper: Autocatalytic processing of the TPR Thiol Protease from Porphyromonas gingivalis

TitleAutocatalytic processing of the TPR Thiol Protease from Porphyromonas gingivalis
Authors
Issue Date2015
PublisherSage Publications, Inc. The Journal's web site is located at http://jdr.sagepub.com/
Citation
Journal of Dental Research, v. 94 Spec. Iss. A, abstract no. 4429 How to Cite?
AbstractObjectives: 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)
Persistent Identifierhttp://hdl.handle.net/10722/225003
ISSN
2015 Impact Factor: 4.602
2015 SCImago Journal Rankings: 1.714

 

DC FieldValueLanguage
dc.contributor.authorHuo, Y-
dc.contributor.authorZhang, Q-
dc.contributor.authorBartlam, M-
dc.contributor.authorWatt, RM-
dc.date.accessioned2016-04-18T03:35:15Z-
dc.date.available2016-04-18T03:35:15Z-
dc.date.issued2015-
dc.identifier.citationJournal of Dental Research, v. 94 Spec. Iss. A, abstract no. 4429-
dc.identifier.issn0022-0345-
dc.identifier.urihttp://hdl.handle.net/10722/225003-
dc.description.abstractObjectives: 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)-
dc.languageeng-
dc.publisherSage Publications, Inc. The Journal's web site is located at http://jdr.sagepub.com/-
dc.relation.ispartofJournal of Dental Research-
dc.rightsJournal of Dental Research. Copyright © Sage Publications, Inc.-
dc.titleAutocatalytic processing of the TPR Thiol Protease from Porphyromonas gingivalis-
dc.typeConference_Paper-
dc.identifier.emailWatt, RM: rmwatt@hku.hk-
dc.identifier.authorityWatt, RM=rp00043-
dc.identifier.hkuros257450-
dc.identifier.volume94-
dc.identifier.issueSpec. Iss. A-
dc.publisher.placeUnited States-

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