Universität zu Köln

Pichlo, Christian, Montada, Angelika A., Schacherl, Magdalena ORCID: 0000-0002-5478-2509 and Baumann, Ulrich (2016). Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD. J. Vis. Exp. (118). CAMBRIDGE: JOURNAL OF VISUALIZED EXPERIMENTS. ISSN 1940-087X

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Abstract

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Pichlo, Christian UNSPECIFIED UNSPECIFIED UNSPECIFIED Montada, Angelika A. UNSPECIFIED UNSPECIFIED UNSPECIFIED Schacherl, Magdalena UNSPECIFIED orcid.org/0000-0002-5478-2509 UNSPECIFIED Baumann, Ulrich UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-254925
DOI:	10.3791/55022
Journal or Publication Title:	J. Vis. Exp.
Number:	118
Date:	2016
Publisher:	JOURNAL OF VISUALIZED EXPERIMENTS
Place of Publication:	CAMBRIDGE
ISSN:	1940-087X
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CLOSTRIDIUM-DIFFICILE; INFECTION; DISCOVERY; EPIDEMIC; CLEAVAGE; SYSTEM; PHENIX; ZMP1 Multiple languages Multidisciplinary Sciences Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/25492