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Freestyle293 cells

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FreeStyle293 cells are a human embryonic kidney (HEK) cell line designed for high-yield protein production in suspension culture. These cells are adapted for growth in serum-free media and are suitable for transient transfection, facilitating the rapid generation of recombinant proteins.

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Lab products found in correlation

Anti flag m2 antibody affinity gel, by Merck Group (8 mentions) Hiload 16 600 superdex 200 pg, by GE Healthcare (1 mentions) Strep tactin resin, by IBA Lifesciences (1 mentions) 293fectin, by Thermo Fisher Scientific (1 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions) Gene fragments, by Integrated DNA Technologies (1 mentions) Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions) Codon optimization tool, by Integrated DNA Technologies (1 mentions) Glutathione agarose 4b, by GE Healthcare (1 mentions) Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions) Expicho expression medium, by Thermo Fisher Scientific (1 mentions) Ht supplement, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Expicho s cells, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Biowest (1 mentions) Fetal bovine serum (fbs), by Biowest (1 mentions)

14 protocols using freestyle293 cells

1

Preparation of Human Protein Library

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In order to prepare human protein library, the CMV promoter-based vectors encoding a cDNA fragment of human gene (~14,000) was transiently transfected into FreeStyle293 cells (Invitrogen) using 293fectin (Invitrogen) according to the manufacturer's instructions. After 3 days of incubation, cells were disrupted in a bead beater and the cell disruptions were frozen at −80 °C until use.
To prepare the membrane-associated human GLUT8, the pcDNA3.1 vector (Invitrogen) encoding a cDNA fragment of human GLUT8 was transiently transfected into FreeStyle293 cells. After 3 days of incubation, cells were washed with phosphate-buffered saline (PBS) and suspended in buffer A, which contained 50 mM HEPES (pH 7.4), 1 mM EDTA. The cells were homogenized and centrifuged at 950×g for 10 min at 4 °C, after which the supernatant was recovered. Total membrane fractions were isolated by ultracentrifugation at 140,000×g for 60 min at 4 °C, and resuspended in buffer A. Membrane fractions were frozen at −80 °C until use.
McMillan E.A., Ryu M.J., Diep C.H., Mendiratta S., Clemenceau J.R., Vaden R.M., Kim J.H., Motoyaji T., Covington K.R., Peyton M., Huffman K., Wu X., Girard L., Sung Y., Chen P.H., Mallipeddi P.L., Lee J.Y., Hanson J., Voruganti S., Yu Y., Park S., Sudderth J., DeSevo C., Muzny D.M., Doddapaneni H., Gazdar A., Gibbs R.A., Hwang T.H., Heymach J.V., Wistuba I., Coombes K.R., Williams N.S., Wheeler D.A., MacMillan J.B., Deberardinis R.J., Roth M.G., Posner B.A., Minna J.D., Kim H.S, & White M.A. (2018). Chemistry-first approach for nomination of personalized treatment in lung cancer. Cell, 173(4), 864-878.e29.
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2

Engineered Mouse PlexinA1/A2 Sema Domain Protein

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Example 22

A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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3

Recombinant CCHFV GP38 Protein Production

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A gene fragment encoding residues 1 to 515 of CCHFV strain IbAr 10200 GPC was codon optimized for human cell expression (via GenScript) and cloned into a pαH eukaryotic expression plasmid with a C-terminal HRV3C protease cleavage site, an 8× HisTag, and a Twin-Strep-tag. This plasmid was transiently transfected into FreeStyle 293 cells (Invitrogen) using polyethyleneimine. Transfected FreeStyle 293 cells were treated with 5 μM kifunensine to ensure uniform high-mannose glycosylation. GP38 was expressed in soluble form and secreted out into the medium, which was harvested and purified over Strep-Tactin resin (IBA Lifesciences). A furin cleavage site (residues 244 to 247) separates the N-terminal MLD and GP38 protein. Because not all of the MLD was cleaved from GP38 by endogenous furin, additional MLD was cleaved off by treatment with furin protease during Strep-tag-based affinity purification. After elution of GP38 from Strep-Tactin resin, the affinity tags were removed by HRV3C cleavage. GP38 was further purified by SEC using a HiLoad 16/600 Superdex 200 pg (GE Healthcare Biosciences) in 2 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% NaN3.
Mishra A.K., Moyer C.L., Abelson D.M., Deer D.J., El Omari K., Duman R., Lobel L., Lutwama J.J., Dye J.M., Wagner A., Chandran K., Cross R.W., Geisbert T.W., Zeitlin L., Bornholdt Z.A, & McLellan J.S. (2020). Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38. Journal of Virology, 94(8), e02005-19.
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4

Stabilized HIV-1 gp140 Env Trimer Production

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Recombinant gp140 Envs were expressed as chimeric SOSIPs, where the gp120 of CH0848 was grafted onto the BG505 gp41 [88 (link)]. The trimers were stabilized with E64K and A316W mutations [15 (link), 49 (link)]. Proteins were expressed in Freestyle293 cells (Invitrogen) using transient cotransfection of Env and furin DNA. Cell-free culture media was subjected to PGT151 antibody affinity and superdex200 size exclusion chromatography.
Francica J.R., Laga R., Lynn G.M., Mužíková G., Androvič L., Aussedat B., Walkowicz W.E., Padhan K., Ramirez-Valdez R.A., Parks R., Schmidt S.D., Flynn B.J., Tsybovsky Y., Stewart-Jones G.B., Saunders K.O., Baharom F., Petrovas C., Haynes B.F, & Seder R.A. (2019). Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates. PLoS Biology, 17(6), e3000328.
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5

Recombinant CCHFV GP38 Protein Production

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Recombinant CCHFV GP38 proteins were produced from the following isolates: Oman-199809166 (UniProt: A0A0U3C6Q7), Kosova-Hoti (UniProt: B2BSL7), 200406546-Turkey (UniProt: A0A0U2SQZ0), Afg09–2990 (UniProt: E5FEZ4), and 79121M18 (UniProt: D4NYK3). Gene fragments (Integrated DNA Technologies) of each isolate’s MLD-GP38 sequence encoding for residues 1–515, as denoted by CCHFV IbAr10200 strain GPC numbering, were codon-optimized for human cell expression (GenScript Codon Optimization Tool). Gene fragments were each cloned into a pαH eukaryotic expression vector with a C-terminal HRV 3C protease cleavage site, an 8x HisTag, and a Twin-Strep-tag. The plasmid for CCHFV strain IbAr10200 GP38 was previously reported28 (link). To ensure cleavage of the MLD from GP38, a pCDNA3.1 plasmid encoding for furin was co-transfected with each clinical GP38 plasmid at a mass ratio of 1:9 furin:GP38. The two plasmids were transiently transfected into FreeStyle 293 cells (Invitrogen) using polyethylenimine followed by treatment with 5 μM kifunensine to ensure uniform high-mannose glycosylation. Soluble GP38 was secreted from the harvested medium and purified via Ni-NTA resin (Thermo Scientific HisPur™ Ni-NTA Resin). GP38 proteins were further purified via SEC using a Superdex 200 Increase 10/300 GL (GE Healthcare Biosciences) in 2 mM Tris pH 8.0, 200 mM NaCl, and 0.02% NaN3.
Shin O.S., Monticelli S.R., Hjorth C.K., Hornet V., Doyle M., Abelson D., Kuehne A.I., Wang A., Bakken R.R., Mishra A., Middlecamp M., Champney E., Stuart L., Maurer D.P., Li J., Berrigan J., Barajas J., Balinandi S., Lutwama J.J., Lobel L., Zeitlin L., Walker L.M., Dye J.M., Chandran K., Herbert A.S., Pauli N.T, & McLellan J.S. (2024). Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38. bioRxiv.
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6

Soluble mouse PlexinA1 protein production

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Example 4

Soluble mouse PlexinA1 protein was designed on the basis of the amino acid sequence of NCBI Reference Sequence NP_032907.1 (SEQ ID NO: 52) up to the extracellular domain. The signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag sequence (SEQ ID NO: 5) was inserted at the C-terminus. The prepared amino acid sequence is shown in (SEQ ID NO: 16). The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the soluble mouse PlexinA1 protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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7

Production of Mouse PlexinA1 Sema Domain

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Example 20

A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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8

Designing Chimeric Plexin Sema Domains

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Example 22

A mouse PlexinA1/A2 sema domain chimeric protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and a sequence from arginine at position 459 to serine at position 510 of the mouse PlexinA2 sema domain protein, the FLAG tag (SEQ ID NO: 5), and the termination codon were added to a sequence after isoleucine at position 458 of the mouse PlexinA1 sema domain protein. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 40. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1/A2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US11576969B2. AntiPlexin A1 agonist antibody (2023-02-14). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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9

Recombinant Mouse PlexinA1 Sema Domain

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Example 20

A mouse PlexinA1 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP_032907.1 to encode a sequence in which the signal peptide (from the N-terminus to isoleucine at position 24) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 512. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 38. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA1 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US11576969B2. AntiPlexin A1 agonist antibody (2023-02-14). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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10

Purification of Mouse PlexinA2 Sema Domain

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Example 21

A mouse PlexinA2 sema domain protein gene was designed on the basis of the sequence of NCBI Reference Sequence NP 032908.2 (SEQ ID NO: 53) to encode a sequence in which the signal peptide (from the N-terminus to glycine at position 31) was replaced with artificial signal peptide HMM+38 (SEQ ID NO: 7), and the FLAG tag (SEQ ID NO: 5) and the termination codon were added after serine at position 510. This gene was prepared by gene synthesis. The amino acid sequence is shown in SEQ ID NO: 39. The prepared gene was incorporated into an expression vector and then introduced into FreeStyle293 cells from Invitrogen for expression, and the mouse PlexinA2 sema domain protein was purified from the culture supernatant by affinity purification using anti-FLAG M2 antibody affinity gel (Sigma-Aldrich) and gel filtration chromatography.

US10688178B2. AntiPlexin A1 agonist antibody (2020-06-23). OSAKA UNIVERSITY [JP], CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Atsushi Kumanogoh [JP], Ryusuke Omiya [JP], Hiroyuki Tsunoda [JP], Takeshi Baba [JP], Sachiyo Suzuki [JP], Yuri Teranishi [JP].
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