For initial in vivo and ex vivo capping, cell pellets from 1 L pHT1T2T3 cultures were lysed using BPER-II amended with recombinant lysozyme and benzonase according to manufacturer’s recommendation and clarified by centrifugation as above. Shell lysates were then mixed with clarified PSII lysates (2:1 v/v ratio of shell to pentamer pellets for HT1T2T3 shells; 10:1 for all minimal shells; see Supplementary Methods/Expression and purification of PSII protein) and incubated for 30 min to allow capping to occur. Lysates were then applied to a 5 mL StrepTrap (GE Healthcare) column equilibrated with Buffer A (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA). The column was washed with six column volumes Buffer A and proteins were eluted in six column volumes Buffer B (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 2.5 mM D-desthiobiotin). Where specified, shells were further purified with anion-exchange chromatography and/or concentrated with 100 kDa molecular weight cut-off Amicon spin filters as described. Capping experiments involving encapsulated cargo (Figs. 4 , 5 ) were scaled down to 0.2 L shell cultures and purified with 1 mL StrepTrap columns using otherwise identical methods.
Streptrap
Manufactured by GE Healthcare
StrepTrap is a laboratory instrument designed for the rapid and efficient capture and purification of streptavidin-tagged proteins. The device utilizes a proprietary resin-based system to selectively bind and isolate the target proteins from complex biological samples. StrepTrap is a versatile tool suitable for a wide range of applications in protein research, biochemistry, and biotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Histrap, by GE Healthcare (3 mentions)
Esf 921 media, by Expression Systems (2 mentions)
Speci c antibodies, by Merck Group (2 mentions)
Ppicza, by Thermo Fisher Scientific (2 mentions)
Top10f bacterial competent cells, by Thermo Fisher Scientific (2 mentions)
Superdex 16 600 200 pg, by GE Healthcare (2 mentions)
Sf9 insect cells, by Expression Systems (2 mentions)
Amicon ultrafiltration unit, by Merck Group (1 mentions)
Superose 6 10 300 column, by GE Healthcare (1 mentions)
Superose 6 column, by GE Healthcare (1 mentions)
25 kda linear polyethylenimine, by Polysciences (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
9 protocols using streptrap
1
In Vivo and Ex Vivo Capping of Protein Shells
2
Reconstitution of the Smc5-6 Heterodimer
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To reconstitute the FL Smc5-6 heterodimer, individual subunits were expressed and
purified as previously described22 (link)23 (link), with the exception that
we used a StrepTrap® column (GE Healthcare) instead of
StrepTactin®. After the StrepTrap® chromatograpy, we
mixed 5 mL of the Smc6 eluate with 150 µL of the Smc5 eluate, and
supplemented this mixture with phosphatase and protease inhibitor cocktail set
IV (EMD). After overnight incubation at 4 °C, the proteins were
concentrated via ultrafiltration using Amicon Ultra filtration units (10K NMWL;
Millipore) and complexes were separated from monomeric subunits by size
exclusion chromatography on a Superose 6 10/300 column (GE Healthcare) in buffer
G (25 mM K2HPO4/KH2PO4, pH 8.0, 750
mM NaCl, 15% glycerol, 0.2% Tween 20, 2 mM 2-ME, 1 mM EDTA). The final fractions
containing purified proteins were concentrated to ~150 ng/mL with Amicon Ultra
filtration units (10K NMWL; Millipore), frozen on dry ice and stored at
−80 °C.
purified as previously described22 (link)23 (link), with the exception that
we used a StrepTrap® column (GE Healthcare) instead of
StrepTactin®. After the StrepTrap® chromatograpy, we
mixed 5 mL of the Smc6 eluate with 150 µL of the Smc5 eluate, and
supplemented this mixture with phosphatase and protease inhibitor cocktail set
IV (EMD). After overnight incubation at 4 °C, the proteins were
concentrated via ultrafiltration using Amicon Ultra filtration units (10K NMWL;
Millipore) and complexes were separated from monomeric subunits by size
exclusion chromatography on a Superose 6 10/300 column (GE Healthcare) in buffer
G (25 mM K2HPO4/KH2PO4, pH 8.0, 750
mM NaCl, 15% glycerol, 0.2% Tween 20, 2 mM 2-ME, 1 mM EDTA). The final fractions
containing purified proteins were concentrated to ~150 ng/mL with Amicon Ultra
filtration units (10K NMWL; Millipore), frozen on dry ice and stored at
−80 °C.
3
Ligation of Dcp1/Dcp2 Proteins
4
Affinity Purification of Protein
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The supernatant was filtered and loaded onto StrepTrap (GE Healthcare) columns previously equilibrated with Tris-HCl buffer (100 mM Tris-HCl pH 8.0, 300 mM KCl, 5% glycerol and 25 mM MgCl2). The columns were washed with the same buffer until the UV baseline reached zero. The elution was performed with 2.5 mM desthiobiotin supplemented equilibration buffer. Selected fractions were pooled together and concentrated using 30 kDa amicon filters and flash frozen in liquid nitrogen.
5
Expression and Purification of MOKV G Proteins
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As explained in Backovic and Krey [48 (link)], stable cell lines expressing the MOKV G1-436WT or MOKV G1-436* proteins with a C-terminal tandem StrepTag were generated by cotransfecting S2 cells with a plasmid encoding MOKV G1-436WT or MOKV G1-436* in a pT350 vector and a plasmid encoding the puromycin resistance gene. To produce MOKV G1-436WT or MOKV G1-436* in large quantities, the S2 cells were grown in suspension at 28°C and 120 rpm until reaching 1.5 107 cells/ml in media supplemented with 7μg/ml puromycin. The cell culture was then diluted twice and induced with 500μM of CuSO4. The culture was harvested five days after induction, and the supernatant was concentrated through tangential filtration (Vivaflow 200, VWR). MOKV G1-436WT or MOKV G1-436* were purified thanks to their StrepTag on a streptavidin affinity column (StrepTrap, GE Healthcare) in a 20 mM Tris-HCl pH 8 buffer, 150 mM NaCl, and 2 mM EDTA. Elution was carried out in the same buffer supplemented with 3 mM α-Desthiobiotin. For MOKV G1-436*, an additional purification step was performed on a size exclusion chromatography (Superdex 200 increase column 10/300, GE Healthcare) equilibrated in 20mM Tris-HCl pH 8, 150mM NaCl, and 2mM EDTA. Purified MOKV G1-436WT or MOKV G1-436* were concentrated (Amicon Ultra 30kDa c / o -Millipore-) and stored at -80°C.
6
Recombinant Protein Expression and Purification
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SC proteins and variants were expressed in HEK293-6E cells (NRC Biotechnology Research Institute) transiently transfected with plasmid DNA using 25 kDa linear polyethylenimine (Polysciences; Warrington, PA). Six days following transfection, cell culture supernatants were subjected to affinity chromatography, either StrepTrap or HisTrap (GE Healthcare Bio-Sciences; Pittsburgh,PA), and further purified by SEC using a Superdex 200 column (GE Healthcare). Purifications were conducted in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) supplemented with either 2.5 mM desthiobiotin or 250 mM imidazole for StrepTrap or HisTrap elution.
Human pIgA was provided by J. Vaerman (Catholic University of Louvain, Brussels, Belgium) (Vaerman et al., 1995 (link)) and further purified by SEC using a Superose 6 column (GE Healthcare) to isolate dIgA. Human pIgM was purchased from Sigma-Aldrich and further purified by SEC, as described for pIgA, to isolate pentameric IgM.
Human pIgA was provided by J. Vaerman (Catholic University of Louvain, Brussels, Belgium) (Vaerman et al., 1995 (link)) and further purified by SEC using a Superose 6 column (GE Healthcare) to isolate dIgA. Human pIgM was purchased from Sigma-Aldrich and further purified by SEC, as described for pIgA, to isolate pentameric IgM.
7
Purification of Recombinant Proteins
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The vector pPICZ-A and Top10F' bacterial competent cells were from Invitrogen and speci c antibodies were from Millipore and Cell Signaling. SF9 insect cells and ESF 921 media were from Expression systems. Strep-Trap, His-Trap and gel ltration columns (Superdex 16/600 200 pg) were from GE. All other chemicals unless otherwise stated were from Sigma Aldrich.
8
Ligation of Dcp1/Dcp2 Proteins
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Dcp1/Dcp2(1–266) containing a C-terminal LPETGGH S. aureus SortaseA recognition site and labeled at ILVMA terminal methyl groups was expressed and purified as described above. Purified protein was then mixed with at least five-fold molar excess His6-MBP-G3-Dcp2(274–504)-StrepII expressed in LB medium and purified using GE StrepTrap and Q ion exchange columns. eSrtA equal to 0.5 molar equivalent of Dcp1/Dcp2(1–266) and 2 mM CaCl2 were added to the solution prior to initiation of the reaction with TEV protease, which cleaves the His6-MBP tag from G3-Dcp2(274–504)-StrepII to expose the required N-terminal glycine for ligation. The reaction was dialyzed against 25mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl2 overnight at 4°C57 (link). Ligated Dcp1/Dcp2ext, containing a mutational scar from (267STAPSDL273 in wild-type to 267LPETGGG273), was purified by heparin and StrepTrap chromatography as described. A final dialysis in 25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM DTT was performed overnight at 4°C prior to NMR experiments.
9
Purification of Recombinant Proteins
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The vector pPICZ-A and Top10F' bacterial competent cells were from Invitrogen and speci c antibodies were from Millipore and Cell Signaling. SF9 insect cells and ESF 921 media were from Expression systems. Strep-Trap, His-Trap and gel ltration columns (Superdex 16/600 200 pg) were from GE. All other chemicals unless otherwise stated were from Sigma Aldrich.
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