HSA fusion proteins were produced by transient transfection of HEK293E cells. Transfected cells were grown in serum-free 293Freestyle media (Gibco, #12338018). Secreted protein was purified from supernatant using CaptureSelect Human albumin affinity matrix (ThermoFisher, #191297005) by elution with 2 M MgCl2, pH 7.4. Purified protein was concentrated and buffer exchanged into PBS by Vivaspin2 centrifugal concentrators (Sartorius). The bispecific LiTE protein was expressed and purified by Nickel-NTA followed by protein A affinity chromatography, as described elsewhere26 (link). For size exclusion chromatography the protein sample in 100 mM Tris-Hcl pH 7.4 was run using a Yarra™ 1.8 µm SEC-X150 column (Phenomenex) on a Dionex Ultimate 3000 HPLC (ThermoFisher Scientific) with a flowrate of 0.1 mL per minute. A protein standard ranging from 670 kDa to 0.244 kDa (Phenomenex) was run with the same settings and overlaid for comparison.
293freestyle media
Manufactured by Thermo Fisher Scientific
Sourced in Canada
293Freestyle media is a serum-free, chemically defined cell culture medium designed for high-density transient transfection of suspension-adapted human embryonic kidney (HEK) 293-derived cell lines. The medium supports rapid cell growth and high recombinant protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Medta, by Merck Group (2 mentions)
Dionex ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Yarra 1.8 m sec x150 column, by Phenomenex (1 mentions)
Captureselect human albumin affinity matrix, by Thermo Fisher Scientific (1 mentions)
Vivaspin2 centrifugal concentrators, by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Jurkat lucia nfat cd16 cells, by InvivoGen (1 mentions)
4 protocols using 293freestyle media
1
Purification of Bispecific Fusion Proteins
2
Culturing HEK293F, HEK293T, and CEM-NK^R_CCR5 Cells
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HEK293F suspension cells were cultured in 293Freestyle media (GIBCO) and grown in a shaking incubator at 37°C, 5% CO2, 70% humidity at 125rpm. HEK293T cells were obtained from Dr. George Shaw (University of Alabama, Birmingham, AL) and were used for the expression of proviral IMC plasmid DNA for ADCC assays. These adherent cell lines were cultured at 37°C, 5% CO2, in DMEM containing 10% heat-inactivated fetal bovine serum (GIBCO) and supplemented with 50 μg/ml gentamicin (Sigma). Cells were disrupted at confluence with 0.25% trypsin in 1 m MEDTA (Sigma) every 48–72 hours. CEM-NKRCCR5 were obtained from the AIDS Reagent Program (Division of AIDS, NIAID, NIH) and used as target cells in the ADCC assay. These cells were cultured at 37°C, 5% CO2 in RPMI containing 10% heat-inactivated fetal bovine serum (GIBCO) and 1% Penicillin Streptomycin (GIBCO). TZM-bl cells (JC53-bl (clone 13)) cells were obtained from AIDS Reagent Program (Division of AIDS, NIAID, NIH) and used for the neutralization assays (Montefiori, 2005 ).
3
Culturing HEK293F, HEK293T, and CEM-NK^R_CCR5 Cells
4
Cell Culture Conditions for HEK293 and Immune Cells
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HEK293F suspension cells were cultured in 293 Freestyle media (Gibco BRL Life Technologies, Ontario, CA) and incubated shaking at 37°C, 5% CO2 and 70% humidity at 125 rpm. HEK293T cells were cultured at 37°C, 5% CO2, in DMEM (Gibco BRL Life Technologies, Ontario, CA) containing 10% heat-inactivated fetal bovine serum (FBS) and supplemented with 50 μg/ml Gentamicin. Cells were disrupted at confluence with 0.25% trypsin in 1 mM EDTA every 48h–72h. HEK293T/ACE2.MF cells were maintained as for HEK293T cells but were supplemented with 3 μg/ml Puromycin for selection of stably transduced cells. THP-1 cells were used for the ADCP assay and obtained from the AIDS Reagent Program, Division of AIDS, NIAID, NIH contributed by Dr. Li Wu and Vineet N. Kewal Ramani. Cells were cultured at 37°C, 5% CO2 in RPMI (Gibco BRL Life Technologies, Ontario, CA) containing 10% heat-inactivated FBS with 1% Penicillin-Streptomycin (Pen/Strep) and 2-mercaptoethanol to a final concentration of 0.05 mM and not allowed to exceed 4 × 105 cells/ml to prevent differentiation. Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, USA) were maintained in IMDM (Gibco BRL Life Technologies, Ontario, CA) media with 10% heat-inactivated FBS, 1% Pen/Strep, and 10 μg/ml of Blasticidin and 100 μg/ml of Zeocin were added to the growth medium every second passage to allow the selection of CD16 expressing cells.
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