Alpha prefoldin, beta prefoldin and the mixture were loaded onto a Superdex S200 Increase 10/300 GL column using an AKTA start, and separated by SEC. Size Exclusion Chromatography was kindly performed by Ms Hélène Lebhar. IMAC purified alpha prefoldin and beta prefoldin were mixed in a 1:2 molar ratio to a total volume of 1 mL at concentrations of 1 mg/mL in PBS pH 8 and incubated overnight at 4☌. SEC was used to demonstrate the formation of aPFD and bPFD hexamers, and to investigate of the monodispersity of the sample. Size Exclusion Chromatography (SEC) is an appropriate method for analysing the assembly of prefoldin hexamers as it retains the native structure of assemblies and can distinguish between molecules of varying size. Successful purification of 6xHis bPFD-SnoopCatcher confirmed (MW: 28 kDa). Lanes are labelled as flow through (FT), wash (W) and elutions (E1, E2, E3). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard. The 6xHis bPFD-SnoopCatcher was successfully purified as reflected by the clear bands seen at the expected molecular weight range (28 kDa) in the elution lanes.įigure 2: SDS-PAGE analysis of IMAC purification of 6xHis bPFD-SnoopCatcher (BBa_K2710003). The purification was analysed by SDS-PAGE (Figure 2). coli T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The part was validated by sequence verification and a diagnostic gel.īBa_K2710003 (encoding 6xHis bPFD-SnoopCatcher) was subcloned into the multiple cloning site of pET19b for expression in E. The Spy and Snoop attachment systems are orthogonal, as SnoopTag/SnoopCatcher does not interact with SpyTag/Sp圜atcher 2.Ī 6x HisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification 4. Similar in function to the Sp圜atcher/Tag system 3, fusion of the SnoopCatcher and SnoopTag to different proteins enables their spontaneous covalent conjugation within minutes. The SnoopCatcher and SnoopTag system was derived from the adhesin RrgA from Streptococcus pneumoniae 2. The hexamer is stable at high temperatures, with a Tm ≥ 70☌ 1.įigure 1: aPFD (red) and bPFD (pink) form hexamers. Each subunit contains flexible alpha-helical coiled coils, 60 to 70 Å in length, which extend from the beta barrel platform 1. The prefoldin hexamer is assembled through interactions between beta hairpins in each subunit, whereby the beta hairpins form two 8 stranded up and down beta barrels 1. Alpha prefoldin (15.7 kDa) and beta prefoldin (13.8 kDa) derived from the thermophilic arcahea Methanobacterium thermoautotrophicum self assemble into an 87 kDa hexamer. Alpha prefoldin (aPFD) and beta prefoldin (bPFD) are two subclasses of prefoldin which oligomerise to form a heterohexameric structure consisting of two alpha subunits and four beta subunits 1(Figure 1). Prefoldin is a molecular chaperone protein which assists in the correct folding of nascent proteins 1. A SnoopCatcher and (GSG)x3 linker was added to the C-terminus. A HisTag and GSG linker was added to the N-terminus of the protein beta prefoldin.
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