Restriction cloning was used to insert the ORF from pQE-ZZ-RanQ69L (Nachury and Weis, 1999 (link)) between the BamH1 and HindIII sites in pRSET A, resulting in pRSET ZZ-RanQ69L. The pRSET zzRCC1 was created by inserting the PCR-amplified wild-type (WT) human RCC1 C-terminally of the ZZ-tag in pRSET A. Site-directed mutagenesis and PCR cloning were used to modify Rango-2 (Kaláb and Soderholm, 2010 (link)) by removing the KPN1 sites from YPet and CyPet (Nguyen and Daugherty, 2005 (link)) and replacing the Snurportin-1 IBB with the IBB amplified from human importin α1 (KPNA2). While doing so, the IBB-importin α1 domain was inserted either with (pK44) or without (pK188) flexible GGCGG linkers added between the 5’ and 3’ ends of IBB and the fluorophores. Restriction cloning was used to combine the C-terminal biotin acceptor peptide tag Avitag (GLNDIFEAQKIEWHE) from pAC-6 (Avidity, Aurora, CO) with WT human importin β (Chi et al., 1997 (link)) in pRSET A vector, resulting in in pRSET importin β-Avitag (pKW1982; pK1099). Restriction cloning in the modified pRSET A with C-terminal Avitag was used to create pRSET-EGFP-Avitag (pK803). The pGEX-2TK1 plasmid for the expression of the S. cerevisiae Nsp1(497-608) FxFG domain was obtained from M. Rexach (Yamada et al., 2010 (link)).
Avitag
Manufactured by Avidity
Sourced in United States
The AviTag is a small peptide tag that can be fused to a protein of interest. The AviTag serves as a substrate for the enzyme biotin ligase, which can covalently attach a biotin molecule to the tag. This allows for the specific labeling and detection of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200, by GE Healthcare (3 mentions)
Avitag, by GenScript (2 mentions)
Pierce biotin quantification kit, by Thermo Fisher Scientific (2 mentions)
Streptavidin conjugated apc fluorophores, by BioLegend (2 mentions)
Pe conjugated streptavidin, by BioLegend (2 mentions)
Bira biotin protein ligase reaction kit, by Avidity (2 mentions)
Ni nta agarose resin, by Qiagen (2 mentions)
Bl21 de3 cell line, by New England Biolabs (2 mentions)
Microfluidizer, by Microfluidics (2 mentions)
Baculodirect, by Thermo Fisher Scientific (1 mentions)
Biotin protein ligase bira kit, by Avidity (1 mentions)
Fitc conjugated streptavidin, by BioLegend (1 mentions)
Hitrap protein g hp column, by Cytiva (1 mentions)
Resource s column, by Cytiva (1 mentions)
Lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Sf 900 3 media, by Thermo Fisher Scientific (1 mentions)
Effectene, by Qiagen (1 mentions)
Biacore 8k, by Cytiva (1 mentions)
Ni agarose resin, by Qiagen (1 mentions)
14 protocols using avitag
1
Cloning and Modification of Fusion Proteins
2
Purification and Modification of Kinesin and Tubulin
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The kinesin-1 sequence from Homo sapiens (amino acid 1 to 465) was ligated into the pET30b (Novagen) plasmid containing an N-terminal His6 tag and a C-terminal AviTag (Avidity). The AviTag peptide is covalently linked to biotin by Escherichia coli biotin ligase (BirA) (51 (link)). The plasmid was transformed into an E. coli Rosetta (DE3) pLysS (Novagen). This construct was a gift from Y. Hiratsuka of the School of Materials Science, Japan Advanced Institute of Science and Technology. The His6-tagged kinesin-1 was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity as previously described (52 (link)). The kinesin-14 was constructed from Drosophila melanogaster (amino acids 195 to 700) and cloned into the pBirAcm (Avidity) plasmid containing an N-terminal histidine tag and AviTag. The construct was expressed in BL21 (DE3) Star (Novagen) and purified by Ni-NTA affinity as previously described (34 (link)). Phosphocellulose (PC) tubulin was purified from porcine brains after 2 cycles of assembly-disassembly and PC chromatography (53 (link)) and stored in liquid nitrogen. Fluorophore-tagged tubulin was prepared by adding a 10-fold molar excess of carboxytetramethylrhodamine (TAMRA, C-1171, Invitrogen, Carlsbad, CA, USA) to tubulin for a labeling stoichiometry of 0.40 to 0.70 (54 (link)).
3
Recombinant Protein Production for pHLA-DR1 and LAG-3:Fc
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Soluble pHLA‐DR1 was either refolded from recombinant DR1α and DR1β chains produced in the BL21(DE3) strain of E. coli [29 ], or generated in Spodoptera frugiperda (sf9) insect cells using the BaculoDirect™ expression system (ThermoFisher Scientific) [30 ] as previously described. pHLA‐DR1 molecules were biotinylated by inclusion of a C‐terminal AviTAG™ biotinylation signal sequence on the DR1α chain that was biotinylated using a BirA biotin‐protein ligase kit (Avidity) [36 ]. A fusion protein of the four extracellular domains of LAG‐3 and the Fc domain of IgG (LAG‐3:Fc) was produced in CHO cells as previously described [19 ]. LAG‐3:Fc was stable in a solution of 20 mM sodium citrate, 86 mM sodium chloride, 100 mM l ‐arginine, 0.02% Tween‐20, pH 7.4 with citric acid (TBSB buffer) at concentrations of up to 30 mg/mL.
4
Biotinylated RBD Tetramer Assay
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RBD protein expressed with AviTag was purchased from GenScript. Site-specific biotinylation of the AviTag was performed using BirA Biotin-Protein Ligase Reaction kit (Avidity). Next, unconjugated biotin was removed using Zeba spin desalting columns, 7K MWCO (ThermoFisher). The quantification of reacted biotin was performed using the Pierce Biotin Quantification Kit (ThermoFisher). Biotinylated RBD was incubated with either streptavidin-conjugated PE (Biolegend) or streptavidin-conjugated APC fluorophores (Biolegend) for 20 min on ice at a molar ratio of 4:1 of biotin to streptavidin. Streptavidin-conjugated FITC (BioLegend) was reacted with excess free biotin to form a non-RBD-specific streptavidin probe as a control. Tetramer formation was confirmed using SDS-PAGE gel. Cells were stained for flow cytometry with all three streptavidin probes at the same time as other fluorescent surface markers at a volumetric ratio of 1:100 for RBD-streptavidin-PE and 1:200 for RBD-streptavidin-APC and biotin-streptavidin-FITC.
5
Fab Fragment Production and Purification
6
Bait and Prey Protein Expression
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Bait expression vectors were modified from the pECIA14 vector (Özkan et al., 2013 (link)). An Avitag (Avidity) was added between the hexahistidine and FLAG tags at the C-terminus of the vector with standard cloning procedures to make a new Gateway (Thermo Fisher, Waltham, MA) destination vector. ECD sequences were moved from entry vectors for Beats and Sides, described in (Özkan et al., 2013 (link)), into the modified pECIA14 vector using LR Clonase II (Thermo Fisher). Prey proteins were expressed from the pECIA2 vector (Özkan et al., 2013 (link)).
All proteins, excepting the unpurified prey, were expressed in Drosophila Schneider 2 cells grown in S2 media with 10% fetal bovine serum, 50 units/mL penicillin and 50 μg/mL streptomycin. The unpurified prey proteins were expressed in Sf-900 III media (Thermo Fisher). Proteins were transfected using Effectene (Qiagen, Hilden, Germany), following manufacturer’s instructions. Copper (0.5 mM CuSO4) was added the day after transfection to induce expression of protein. For the baits, 3 mM biotin was also added to the media to facilitate in vivo biotinylation. Prey proteins were purified using Ni-NTA resin, following standard procedures.
All proteins, excepting the unpurified prey, were expressed in Drosophila Schneider 2 cells grown in S2 media with 10% fetal bovine serum, 50 units/mL penicillin and 50 μg/mL streptomycin. The unpurified prey proteins were expressed in Sf-900 III media (Thermo Fisher). Proteins were transfected using Effectene (Qiagen, Hilden, Germany), following manufacturer’s instructions. Copper (0.5 mM CuSO4) was added the day after transfection to induce expression of protein. For the baits, 3 mM biotin was also added to the media to facilitate in vivo biotinylation. Prey proteins were purified using Ni-NTA resin, following standard procedures.
7
Surface Plasmon Resonance Binding Assay
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SPR experiments were performed with a Biacore 8K instrument (Cytiva) using PBST. For binding assays of AiDs with HA-PD1, HA-PD1 with a C-terminal AviTag was biotinylated with BirA according to the manufacturer’s protocol (Avidity) and immobilized on a NeutrAvidin chip (Cytiva, 29407997) to a level of ~250 response units (RUs). Binding measurements were performed using single-cycle kinetics with five 1:3 serial dilutions of each recombinant AiD (at concentrations indicated in figures) using 120 seconds of association and 1200 seconds for the final dissociation time at 25°C and a flow rate of 30 μL/min. The data were analyzed in the same manner as the masked antagonists.
8
In Vivo Protein Biotinylation in E. coli
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The RNase deficient E. coli K-12 strain CAN20/12E (RNase BN−, II−, D−, I−)43 (link) was transformed with two plasmids. The pBirAcm plasmid contained the birA gene overexpressing biotin ligase and the pAN5 plasmid the ribosomal protein uL4 fused to an N-terminal AviTag (14-mer peptide GLNDIFEAQKIEWH) (Avidity, Aurora, USA). The biotin ligase catalyzes transfer of a single biotin molecule specifically at the lysine of the 14-residue AviTag44 (link). Upon induction with 1 mM IPTG at A600 = 0.4 and addition of 50 μM biotin, in vivo biotinylation took place by the biotin ligase. Cells were grown for an additional 1 h at 37 °C and incubated on ice for 1 h before harvesting.
9
Expression and Purification of scTCR Proteins
10
Expression and Purification of scTCR Proteins
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RD1-MART1 and RD1-MART1HIGH were introduced into the pET28a expression vector with a C-terminal AviTag (Avidity) using NcoI and EcoRI restriction sites (forward primer: 5’ - TAT ACC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC AGC AGC GGC CTG GTG CCG CGC GGC AGC aat gct ggt gta aca caa acg cc - 3’, Reverse primer: 5’ - T TTA GAA TTC TTA ttc gtg cca ttc gat ttt ctg agc ctc gaa gat gtc gtt cag acc gcc acc gtc tgg agt gac cac aac ctg ggt - 3’. Plasmids were transformed into the BL21-DE3 cell line (NEB), expanded, and induced for expression. Following induction, cells were passed through a microfluidizer (Microfluidics Corporation, Newton, MA, USA), inclusion bodies were isolated, and protein was purified as previously described64 (link). Soluble scTCR were refolded and purified with Ni-NTA agarose resin (Qiagen, Valencia, CA) followed by gel filtration (Superdex 200, GE Healthcare). Folded scTCRs were biotinylated in vitro (Avidity, BirA enzyme). Biotinylation was verified by gel-shift with streptavidin by SDS-PAGE.
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