Academic Journal
Detection of soluble co-factor dependent protein expression in vivo : Application to the 4′-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis
| العنوان: | Detection of soluble co-factor dependent protein expression in vivo : Application to the 4′-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis |
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| المؤلفون: | Rottier, Karine, Faille, Alexandre, Prudhomme, Thomas, Leblanc, Cécile, Chalut, Christian, Cabantous, Stéphanie, Guilhot, Christophe, Mourey, Lionel, Pedelacq, Jean-Denis |
| المساهمون: | Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Claudius Regaud, ANR-09-BLAN-0298,XPKS-MYCO,Etude de la programmation moléculaire de polykétides synthases de mycobactéries pathogènes(2009) |
| المصدر: | ISSN: 1047-8477. |
| بيانات النشر: | HAL CCSD Elsevier |
| سنة النشر: | 2013 |
| المجموعة: | Université Toulouse III - Paul Sabatier: HAL-UPS |
| مصطلحات موضوعية: | Split-GFP, Domain trapping, Co-factor, 40 -Phosphopantetheinyl transferase, Polyketide, Tuberculosis, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology |
| الوصف: | International audience ; The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4 0-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N-and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg 2+ , essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| Relation: | hal-03002994; https://hal.science/hal-03002994; https://hal.science/hal-03002994/document; https://hal.science/hal-03002994/file/JSB.pdf |
| DOI: | 10.1016/j.jsb.2013.07.010 |
| الاتاحة: | https://hal.science/hal-03002994 https://hal.science/hal-03002994/document https://hal.science/hal-03002994/file/JSB.pdf https://doi.org/10.1016/j.jsb.2013.07.010 |
| Rights: | info:eu-repo/semantics/OpenAccess |
| رقم الانضمام: | edsbas.F15552FC |
| قاعدة البيانات: | BASE |
| DOI: | 10.1016/j.jsb.2013.07.010 |
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