Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases
Summary: Inter-domain movements act as important activity modulators in multi-domain proteins. Here, we present a protocol for inter-domain cross-linking via engineered cysteines. Using collagenase G (ColG) from Hathewaya histolytica as a model, we describe steps for the design, expression, purifica...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723004860 |
| _version_ | 1827862932561592320 |
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| author | Jamil Serwanja Hans Brandstetter Esther Schönauer |
| author_facet | Jamil Serwanja Hans Brandstetter Esther Schönauer |
| author_sort | Jamil Serwanja |
| collection | DOAJ |
| description | Summary: Inter-domain movements act as important activity modulators in multi-domain proteins. Here, we present a protocol for inter-domain cross-linking via engineered cysteines. Using collagenase G (ColG) from Hathewaya histolytica as a model, we describe steps for the design, expression, purification, and cross-linking of the target protein. We detail a system to monitor the progress of the cross-linking reaction and to confirm the structural integrity of the purified cross-linked proteins. We anticipate this protocol to be readily adaptable to other multi-domain enzymes.For complete details on the use and execution of this protocol, please refer to Serwanja et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-12T14:01:38Z |
| format | Article |
| id | doaj.art-3ef7787eceb547838eaef986d5aa924c |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-12T14:01:38Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-3ef7787eceb547838eaef986d5aa924c2023-08-22T04:07:00ZengElsevierSTAR Protocols2666-16672023-09-0143102519Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenasesJamil Serwanja0Hans Brandstetter1Esther Schönauer2Department of Biosciences and Medical Biology, Paris Lodron University of Salzburg, Salzburg 5020, AustriaDepartment of Biosciences and Medical Biology, Paris Lodron University of Salzburg, Salzburg 5020, AustriaDepartment of Biosciences and Medical Biology, Paris Lodron University of Salzburg, Salzburg 5020, Austria; Corresponding authorSummary: Inter-domain movements act as important activity modulators in multi-domain proteins. Here, we present a protocol for inter-domain cross-linking via engineered cysteines. Using collagenase G (ColG) from Hathewaya histolytica as a model, we describe steps for the design, expression, purification, and cross-linking of the target protein. We detail a system to monitor the progress of the cross-linking reaction and to confirm the structural integrity of the purified cross-linked proteins. We anticipate this protocol to be readily adaptable to other multi-domain enzymes.For complete details on the use and execution of this protocol, please refer to Serwanja et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723004860Molecular BiologyProtein BiochemistryStructural Biology |
| spellingShingle | Jamil Serwanja Hans Brandstetter Esther Schönauer Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases STAR Protocols Molecular Biology Protein Biochemistry Structural Biology |
| title | Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases |
| title_full | Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases |
| title_fullStr | Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases |
| title_full_unstemmed | Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases |
| title_short | Quantitative cross-linking via engineered cysteines to study inter-domain interactions in bacterial collagenases |
| title_sort | quantitative cross linking via engineered cysteines to study inter domain interactions in bacterial collagenases |
| topic | Molecular Biology Protein Biochemistry Structural Biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723004860 |
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