ADDRESS Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD UK
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2024 Mark Howarth. All rights reserved.
We have distributed the resources developed in the lab to 45 countries: Please send us a tweet @HowarthSci when you receive one of our reagents Purified Protein Availability We are making purified SpyCatcher003-mi3 VLPs broadly available to the academic community: no need for collaboration but you will need to pay for shipping. Protein for SpyCatcher003, Cys-SpyCatcher003, SpyTag003-MBP and traptavidin are distributed not-for-profit at Kerafast. Certain reagents are also commercialised at the Bio-Rad site. Plasmids The following plasmids were given to the plasmid repository Addgene for easy distribution to academic laboratories. Addgene have shipped at least 2,000 samples from us. Non-academics should contact Mark for information. Feel free to e-mail for comments or advice on these systems. (a) Biotin binding tools (i) Traptavidin, a mutant of streptavidin with lower off-rate, increased thermostability, and greater mechanical strength. Also Traptavidin-E6 (for ion-exchange chromatography purification) (ii) Monovalent streptavidin and cis or trans Divalent streptavidin: Alive-H6 (biotin binding with 6 histidines for Ni-NTA purification) Alive-E6 (biotin binding with 6 glutamates for ion-exchange purification) Dead (streptavidin subunit with no biotin binding) Dead-Aspartate loop (streptavidin subunit with no biotin binding and negatively charged loop for ion-exchange chromatography resolution) (J Mol Biol 2014 and Nature Methods 2006) (iii) SpyAvidin subunits: see below at (b)(iii) (iv) Biotinylation tools pDisplay-BirA-ER for biotinylation in the mammalian secretory pathway pDisplay-AP-CFP-TM as a target for BirA in mammalian cells BirA-His6 for bacterial overexpression (GST-BirA for bacterial overexpression- please request from Chris O’Callaghan) (v) Fluorophore-friendly streptavidin K121R streptavidin or Flavidin plasmids (b) Isopeptide tools See our tips on SpyTag use here. Also, please see a hyperlinked bibliography of all papers using Spy and Snoop tools here. SpyTag003 cloning NOTE! If SpyTag is positioned right next to the initiation codon, with certain codon usage we found low induction in bacteria. This is probably because of secondary structure formation with vector-derived sequences in the mRNA. Including a GSS spacer with the following codons worked well in pET28, with a T7 promoter (download insert sequence): Ideally you should check your particular construct using an online Ribosome Binding Site calculator, with 10,000 representing a satisfactory score. (i) Standard SpyTag/SpyCatcher tools SpyCatcher003 for irreversible peptide-protein ligation SpyCatcher003 S49C for Cysteine-mediated anchoring or labelling SpyCatcher003-sfGFP linked to superfolder GFP SpyTag003-MBP for irreversible peptide-protein ligation AviTag-SpyTag003-MBP combining irreversible ligation via SpyTag003 with a biotinylation tag for anchoring to streptavidin SpyTag003-mKate2 linked to bright red fluorescent protein SpyTag003-mClover3 linked to bright greeen fluorescent protein SpyTag003-sfGFP linked to superfolder GFP SpyTag-sfGFP (original SpyTag linked to superfolder GFP) TfR-sfGFP-myc tag-SpyCatcher003 for mammalian cell-surface display EGFP-Talin head-SpyCatcher003 for mammalian cytosolic expression SpyTag003-Talin rod-mCherry for mammalian cytosolic expression (Also earlier generations of these tools: AviTag-SpyCatcher for anchoring on streptavidin-resin SpyTag-MBP, SpyCatcher, SpyCatcher EQ as a non-reactive control for SpyCatcher MBPx-SpyCatcher for anchoring on amylose-resin SpyCatcher002 for accelerated irreversible peptide-protein ligation AviTag-SpyCatcher002 for anchoring on streptavidin-resin Cys-SpyCatcher002 for thiol-reactive anchoring or labeling SpyTag002-MBP for accelerated irreversible peptide-protein ligation SpyCatcher002 EQ as a non-reactive control) (ii) Ligase and DogCatcher tools AviTag-SnoopLigase for peptide-peptide ligation HaloTag7-SnoopLigase for peptide-peptide ligation SnoopTagJr-MBP for SnoopLigase peptide-peptide ligation or reaction with SnoopCatcher SUMO-DogTag for SnoopLigase peptide-peptide ligation IMX-DogTag for SnoopLigase-mediated oligomerization DogCatcher for ligation to DogTag-partners AviTag-DogCatcher-MBP for anchoring on streptavidin-resin Cys-DogCatcher for thiol-reactive anchoring or labeling DogCatcher-sfGFP sfGFP with SpyTag003 in loop A or loop B or loop C sfGFP with DogTag in loop A or loop B or loop C AviTag-DogTag-MBP for anchoring on streptavidin-resin HaloTag7SS-DogTag SnoopTag2-AffiHER2 for an affibody to HER2 that can be ligated to DogTag2 or SnoopCatcher AviTag-DogTag2-MBP for site-specific biotinylation and ligation to SnoopTag2 or DogCatcher SnoopLigase2 for peptide-peptide ligation TfR-sfGFP-SnoopTag2 for mammalian cell-surface display of SnoopTag2 HA-MBP-SnoopLigase2 N775Q-KDEL for peptide-peptide ligation in the mammalian ER RBD-DogTag2-SpyTag encoding SARS-CoV-2 RBD for reaction with SnoopTag2 or DogCatcher SUMO-DogTag2 HaloTag7-SnoopLigase2 for peptide-peptide ligation SnoopTagJr-sfGFP SnoopTag2-sfGFP (GenBank OQ923256, Addgene 201810) TG2x-DogTag2, a variant of human Transglutaminase 2 for ligation to SnoopTag2 or DogCatcher SnoopTag2-TGFα encoding human Transforming Growth Factor-α for ligation to DogTag2 or SnoopCatcher (SnoopLigase2 or SnoopLigase is recommended over SpyLigase for higher efficiency) (iii) SpyAvidin subunits: Dead-SpyCatcher (DCatch) Dead-SpyTag, (DTag) Traptavidin-E6 (Tre) Traptavidin (Tr) (iv) SpyRings: SpyTag-β-lactamase-SpyCatcher, for enzyme cyclization: through CPEC one can insert other enzymes in this scaffold (v) Solid-phase polyproteam synthesis: SnoopCatcher for irreversible peptide-protein ligation (an orthogonal pair to SpyTag/SpyCatcher) SnoopTag-MBP for irreversible peptide-protein ligation (an orthogonal pair to SpyTag/SpyCatcher) SpyCatcher-SnoopCatcher for bridging SpyTag and SnoopTag MBPx-SpyCatcher for solid-phase anchoring on amylose-resin AviTag-SpyCatcher for solid-phase anchoring on streptavidin-resin SnoopTag-mEGFP-SpyTag, with BamHI sites for easy insertion of other proteins between SnoopTag and SpyTag (vi) Biomaterials: pQE80L TriCatcher-ELP, with two SpyCatchers and one SnoopCatcher pQE80L TriCatcher-RGDSP, as above with an integrin-binding site pQE80L TriCatcher-MMP, as above with an MMP cleavage site pQE80L TriCatcher-RGDSP-MMP, with integrin-binding and MMP cleavage sites pQE80L SpyTag-ELP-SpyTag pQE80L SpyTag-ELP-SpyTag DK (vii) Virus-like particle and oligomer assembly: (see note on protein availability above) pET28a-SpyCatcher-mi3 (with C-tag) pET28a-SpyCatcher-mi3 with His-tag (not described in Bruun et al. but may be more convenient for some labs and we can send Ni- NTA protocol) pET28a-SpyCatcher003-mi3 (with C-tag) IMX-DogTag for SnoopLigase-mediated oligomerization SpyCatcher002-oDi, coiled coil for dimerization SpyCatcher002-oTri, coiled coil for trimerization SpyCatcher002-oTet, coiled coil for tetramerization SpyCatcher002-oPent, coiled coil for pentamerization SpyCatcher002-oHex, coiled coil for hexamerization SpyCatcher002-oHept, coiled coil for heptamerization (vii) Affinity purification: (see note on protein availability above) SpySwitch for capture and release of fusions to SpyTag, SpyTag002 or SpyTag003 SpyDock for capture and release of SpyTag- and SpyTag002-fusions (but SpySwitch is recommended) (viii) Antigens Spy0469 (antigen from Streptococcus pyogenes) PsaA (antigen from Streptococcus pneumoniae) SpyTag003-OspC (antigen from Borrelia burgdorferi; Lyme Disease) SpyTag-RBD (receptor-binding domain from Spike of SARS-CoV-2) NDV HN-SpyTag (antigen from Newcastle Disease Virus) (c) NeissLock OAZ-GSY-SPM ODC-Ctag TGFa-GSY-SPM (Of course, please enquire if there is a useful plasmid on which we have published, that is not listed here.)
Progress in science depends on      new techniques, new discoveries and new ideas,                                        probably in that order ― Brenner