{"id":6767,"date":"2016-03-24T13:39:11","date_gmt":"2016-03-24T12:39:11","guid":{"rendered":"http:\/\/news.embl.de\/?p=6767"},"modified":"2024-03-25T10:28:22","modified_gmt":"2024-03-25T09:28:22","slug":"1603-tuberculosis-tools","status":"publish","type":"post","link":"https:\/\/www.embl.org\/news\/science\/1603-tuberculosis-tools\/","title":{"rendered":"Better tools for exploring TB protein structures"},"content":{"rendered":"\n

Information about the 3D structure of Mtb<\/em> proteins involved in the infection process of tuberculosis can provide crucial clues for designing new drugs. However, Mtb<\/em> proteins are not easy subjects for structural biology studies, and as a result relatively few Mtb<\/em> proteins have been characterised in detail. The need for improved methodology therefore drives the work done within Matthias Wilmanns<\/a>\u2019 group at EMBL in Hamburg. In order to explore the structures of Mtb<\/em> proteins, they have optimised methods for producing the proteins in the laboratory, and now hope to share this knowledge with other structural biologists.<\/p>\n\n\n\n

Scientists have to produce large amounts of Mtb<\/em> proteins in the laboratory before they can be studied. This is done by inserting the protein\u2019s gene into a piece of mobile DNA called a vector. This vector is then shuttled into a bacterium host where it replicates and produces lots of copies of the Mtb<\/em> protein that can subsequently be used for structural studies. Traditionally E.coli<\/em> bacteria have been used as hosts for this purpose, but being so distantly related to Mtb<\/em> this tends to create problems due to, for example, an inability of the protein to fold correctly. \u201cInstead of E. coli<\/em> we use a non-pathogenic close relative of Mtb<\/em> \u2013 Mycobacterium smegmatis<\/em> – which can circumvent expression problems of Mtb<\/em> proteins,\u201d explains Annabel Parret who coordinates the tuberculosis team within the Wilmanns group. \u201cOur eight in-house generated vectors also work with our optimised M. smegmatis<\/i> strain,\u201d she adds.<\/p>\n\n\n\n

Our eight in-house generated vectors work with our optimised M. smegmatis<\/i> strain<\/p><\/blockquote>\n\n\n\n

As well as the gene of interest, vectors include a number of other DNA elements. The \u201cpMy\u201d vector series generated at EMBL enables scientists to choose characteristics best suited to their experiments. These eight vectors offer a combination of different promoters \u2013 stretches of DNA that initiate gene transcription, antibiotic resistance \u2013 used to check that the bacteria has successfully expressed the protein and purification tags\u2013 used to isolate the protein after expression. \u201cAlthough a large collection of vectors exist for E. coli<\/em>, expression tools for M. smegmatis<\/em> are scarce so we hope this versatile toolkit will be helpful for other structural biologists,\u201d says group leader Wilmanns. The new vectors have successfully been used to produce large quantities of a number of Mtb<\/em> proteins that are notoriously difficult to express including membrane and toxic proteins.<\/p>\n\n\n\n

By using multiple vectors simultaneously, different proteins can also be expressed together and quickly screened for their ability to form complexes. \u201cWhile relatively few Mtb<\/em> structures have been characterised, even less is known about which Mtb<\/em> proteins interact with each other and how,\u201d explains Parret. \u201cWe hope these tools will encourage others to also take up studying Mtb<\/em> proteins and move the science that bit faster toward the goal of being able to fight tuberculosis,\u201d concludes Wilmanns.<\/p>\n\n\n\n