title: Tail proteins of phage T5: investigation of the effect of the His6-tag position, from expression to crystallisation.
abstract: Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Marjolaine
authors: M
authors: Noirclerc-Savoye
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Ali
authors: A
authors: Flayhan
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Cindy
authors: C
authors: Pereira
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Benoit
authors: B
authors: Gallet
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Pierre
authors: P
authors: Gans
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France.
authors: Christine
authors: C
authors: Ebel
authors: Univ. Grenoble Alpes, IBS, Grenoble, France; CNRS, UMR5075, IBS, F-38044 Grenoble, France; CEA, DSV, IBS, F-38044 Grenoble, France. Electronic address: Cecile.Breyton@ibs.fr.
authors: Cécile
authors: C
authors: Breyton
mesh_terms: D001435:Bacteriophages / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006639:Histidine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine
keywords: Bacteriophage; Crystallisation; His-tag position; Protein purification