Ni sepharose 6 ff column

Manufactured by Cytiva

Ni Sepharose 6 FF column is a pre-packed affinity chromatography column designed for the purification of histidine-tagged proteins. The column consists of agarose beads coupled with nickel ions, which bind to the histidine tags on the target proteins. This allows for selective capture and purification of the desired proteins from complex mixtures.

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Lab products found in correlation

Akta avant150 fplc system, by Cytiva (8 mentions) Superdex 200 increase 10 300, by Cytiva (7 mentions) Bioflo 320 fermenter, by Eppendorf (7 mentions) Hiload s200 pg gl sec column, by Cytiva (6 mentions) 10k mwco centrifugal filters, by Merck Group (6 mentions) M110p machine, by Microfluidics (1 mentions) Superdex 200 increase 10 300 sec column, by Cytiva (1 mentions)

9 protocols using ni sepharose 6 ff column

Protein Expression and Purification of I53-50A and I53-50B.4PT1

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The I53-50A and I53-50B.4.PT1 proteins were expressed as described recently^{45 (link)}. Briefly, Lemo21 cells(DE3) (NEB), which were grown in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf) were used to express the I53-50A or I53-50B.4PT1 proteins grown. Cells were induced with 1 mM IPTG and maintained shaken for ~16 h at 18 °C. Microfluidization was used to harvest and lyse the cells, using a Microfluidics M110P machine at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Proteins were purified by applying clarified lysates to a 2.6×10 cm Ni Sepharose 6 FF column (Cytiva) on an AKTA Avant150 FPLC system (Cytiva). A linear gradient of 30 mM to 500 mM imidazole in 50 mM Tris, pH 8, 500 mM NaCl, 0.75% CHAPS was used to elute both proteins. Next, the pooled fractions were subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV) whereas I53-50B.4PT1 elutes at ~0.45 CV. Prior to nanoparticle assembly, protein preparations were tested to confirm low levels of endotoxin.

Brouwer P.J., Antanasijevic A., de Gast M., Allen J.D., Bijl T.P., Yasmeen A., Ravichandran R., Burger J.A., Ozorowski G., Torres J.L., LaBranche C., Montefiori D.C., Ringe R.P., van Gils M.J., Moore J.P., Klasse P.J., Crispin M., King N.P., Ward A.B, & Sanders R.W. (2021). Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles. NPJ Vaccines, 6, 24.

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Purification of I53-50A and I53-50B Proteins

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The I53-50A and I53-50B proteins were expressed as described before (Walls et al., 2020b (link)). Briefly, transformed Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) were grown at 37°C to an OD600 ∼0.8 with agitation. Expression was induced with 1 mM IPTG and temperature was reduced to 18°C. Cells were harvested after ∼16 h and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Proteins were eluted with a linear gradient of 30 mM to 500 mM imidazole in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) previously equilibrated in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer.

Tortorici M.A., Walls A.C., Joshi A., Park Y.J., Eguia R.T., Miranda M.C., Kepl E., Dosey A., Stevens-Ayers T., Boeckh M.J., Telenti A., Lanzavecchia A., King N.P., Corti D., Bloom J.D, & Veesler D. (2022). Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein. Cell, 185(13), 2279-2291.e17.

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Production and Purification of I53-50B.4PT1

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Lemo21 (DE3) (NEB) cells expressing I53-50B.4PT1 were grown in a 10L BioFlo 320 Fermenter (Eppendorf) or a 2L shake flask. Cells were grown in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) at 37°C to an OD600 of 0.8. After the cells were induced with 1 mM of IPTG, temperature was reduced to 18°C and cells were grown for 16h. The cells were then lysed in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS using a Microfluidics M110P at 18,000 psi. Lysate was centrifuged at 24,000 g for 30 minutes. Clarified lysate was next applied to a Ni Sepharose 6 FF column (Cytiva) linked to an AKTA Avant150 FPLC system (Cytiva) for immobilized metal affinity chromatography. I53-50B.4PT1 was eluted using a 30 mM to 500mM imidazole linear gradient in 50 mM Tris pH 8, 500 mM NaCl and 0.75% CHAPS. Fractions containing I53-50B.4PT1 were pooled, concentrated using centrifugal filters with a 10,000 kDa cutoff (Millipore), sterilized and applied to a Superdex 200 Increase 10/300 (Cytvia) for further purification. Batches were tested for endotoxin levels before use.

Brouwer P.J., Brinkkemper M., Maisonnasse P., Dereuddre-Bosquet N., Grobben M., Claireaux M., de Gast M., Marlin R., Chesnais V., Diry S., Allen J.D., Watanabe Y., Giezen J.M., Kerster G., Turner H.L., van der Straten K., van der Linden C.A., Aldon Y., Naninck T., Bontjer I., Burger J.A., Poniman M., Mykytyn A.Z., Okba N.M., Schermer E.E., van Breemen M.J., Ravichandran R., Caniels T.G., van Schooten J., Kahlaoui N., Contreras V., Lemaître J., Chapon C., Fang R.H., Villaudy J., Sliepen K., van der Velden Y.U., Haagmans B.L., de Bree G.J., Ginoux E., Ward A.B., Crispin M., King N.P., van der Werf S., van Gils M.J., Le Grand R, & Sanders R.W. (2021). Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell, 184(5), 1188-1200.e19.

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Purification of I53-50A and I53-50B.4PT1 Proteins

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The I53–50A and I53–50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ~0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ~16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6×10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53–50A elutes at ~0.6 column volume (CV). I53–50B.4PT1 elutes at ~0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Fiala B., Schäfer A., Wrenn S., Pham M.N., Murphy M., Tse L.V., Shehata L., O’Connor M.A., Chen C., Navarro M.J., Miranda M.C., Pettie D., Ravichandran R., Kraft J.C., Ogohara C., Palser A., Chalk S., Lee E.C., Kepl E., Chow C.M., Sydeman C., Hodge E.A., Brown B., Fuller J.T., Dinnon KH I.I.I., Gralinski L.E., Leist S.R., Gully K.L., Lewis T.B., Guttman M., Chu H.Y., Lee K.K., Fuller D.H., Baric R.S., Kellam P., Carter L., Pepper M., Sheahan T.P., Veesler D, & King N.P. (2020). Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2. bioRxiv.

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Purification and Characterization of I53-50A and I53-50B.4PT1 Proteins

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The I53-50A and I53-50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV). I53-50B.4PT1 elutes at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Fiala B., Schäfer A., Wrenn S., Pham M.N., Murphy M., Tse L.V., Shehata L., O’Connor M.A., Chen C., Navarro M.J., Miranda M.C., Pettie D., Ravichandran R., Kraft J.C., Ogohara C., Palser A., Chalk S., Lee E.C., Guerriero K., Kepl E., Chow C.M., Sydeman C., Hodge E.A., Brown B., Fuller J.T., Dinnon KH I.I.I., Gralinski L.E., Leist S.R., Gully K.L., Lewis T.B., Guttman M., Chu H.Y., Lee K.K., Fuller D.H., Baric R.S., Kellam P., Carter L., Pepper M., Sheahan T.P., Veesler D, & King N.P. (2020). Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell, 183(5), 1367-1382.e17.

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Purification of Nanoparticle Components

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The nanoparticle components I53-50A and I53-50B.4.PT1,²⁹ (link) and I53_dn5A and I53_dn5B,³⁰ (link) were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) and grown in 2 L baffled shake flasks. Cells were grown at 37°C to an OD600–0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells were shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, (with 0.75% CHAPS only for I53-50 proteins). Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM–500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to a Superdex 200 Increase 10/300 SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Kraft J.C., Pham M.N., Shehata L., Brinkkemper M., Boyoglu-Barnum S., Sprouse K.R., Walls A.C., Cheng S., Murphy M., Pettie D., Ahlrichs M., Sydeman C., Johnson M., Blackstone A., Ellis D., Ravichandran R., Fiala B., Wrenn S., Miranda M., Sliepen K., Brouwer P.J., Antanasijevic A., Veesler D., Ward A.B., Kanekiyo M., Pepper M., Sanders R.W, & King N.P. (2022). Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens. Cell Reports Medicine, 3(10), 100780.

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Production and Purification of I53-50B.4PT1

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Lemo21 (DE3) (NEB) cells expressing I53-50B.4PT1 were grown in a 10 L BioFlo 320 Fermenter (Eppendorf) or a 2 L shake flask. Cells were grown in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) at 37 °C to an OD600 of 0.8. After the cells were induced with 1 mM of IPTG, temperature was reduced to 18 °C and cells were grown for 16 h. The cells were then lysed in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS using a Microfluidics M110P at 18,000 psi. Lysate was centrifuged at 24,000 × g for 30 min. Clarified lysate was next applied to a Ni Sepharose 6 FF column (Cytiva) linked to an AKTA Avant150 FPLC system for immobilized metal affinity chromatography. I53-50B.4PT1 was eluted using a 30–500 mM imidazole linear gradient in 50 mM Tris pH 8, 500 mM NaCl and 0.75% CHAPS. Fractions containing I53-50B.4PT1 were pooled, concentrated using centrifugal filters with a 10,000 kDa cutoff (Millipore), sterilized and applied to a Superdex 200 Increase 10/300 (Cytiva) for further purification. Batches were tested to ensure low levels of endotoxin before use.

Sliepen K., Radić L., Capella-Pujol J., Watanabe Y., Zon I., Chumbe A., Lee W.H., de Gast M., Koopsen J., Koekkoek S., del Moral-Sánchez I., Brouwer P.J., Ravichandran R., Ozorowski G., King N.P., Ward A.B., van Gils M.J., Crispin M., Schinkel J, & Sanders R.W. (2022). Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate. Nature Communications, 13, 7271.

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Expression and Purification of I53-50A and I53-50B.4PT1

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The I53-50A and I53-50B.4.PT1 proteins (Bale et al., 2016 (link)) were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV). I53-50B.4PT1 elutes at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Miranda M.C., Schäfer A., Pham M.N., Greaney A., Arunachalam P.S., Navarro M.J., Tortorici M.A., Rogers K., O’Connor M.A., Shirreff L., Ferrell D.E., Bowen J., Brunette N., Kepl E., Zepeda S.K., Starr T., Hsieh C.L., Fiala B., Wrenn S., Pettie D., Sydeman C., Sprouse K.R., Johnson M., Blackstone A., Ravichandran R., Ogohara C., Carter L., Tilles S.W., Rappuoli R., Leist S.R., Martinez D.R., Clark M., Tisch R., O’Hagan D.T., Van Der Most R., Van Voorhis W.C., Corti D., McLellan J.S., Kleanthous H., Sheahan T.P., Smith K.D., Fuller D.H., Villinger F., Bloom J., Pulendran B., Baric R.S., King N.P, & Veesler D. (2021). Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. Cell, 184(21), 5432-5447.e16.

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Purification of I53-50B.4PT1 and 2obx Proteins

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The I53-50B.4.PT1 and 2obx proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50B.4PT1 and 2obx elute at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

McLeod B., Mabrouk M.T., Miura K., Ravichandran R., Kephart S., Hailemariam S., Pham T.P., Semesi A., Kucharska I., Kundu P., Huang W.C., Johnson M., Blackstone A., Pettie D., Murphy M., Kraft J.C., Leaf E.M., Jiao Y., van de Vegte-Bolmer M., van Gemert G.J., Ramjith J., King C.R., MacGill R.S., Wu Y., Lee K.K., Jore M.M., King N.P., Lovell J.F, & Julien J.P. (2022). Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses. Immunity, 55(9), 1680-1692.e8.

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