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.
Captureselect human albumin affinity matrix
Manufactured by Thermo Fisher Scientific
The CaptureSelect Human albumin affinity matrix is a chromatographic resin designed for the purification of human albumin from various sample sources. Its core function is to selectively bind and capture human albumin, allowing for its efficient separation and isolation.
Automatically generated - may contain errors
Lab products found in correlation
Vivaspin2 centrifugal concentrators, by Sartorius (2 mentions)
Dionex ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
293freestyle media, by Thermo Fisher Scientific (1 mentions)
Yarra 1.8 m sec x150 column, by Phenomenex (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
2 protocols using captureselect human albumin affinity matrix
1
Purification of Bispecific Fusion Proteins
2
Recombinant ACE2 Fusion Protein Production
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The rHA-ACE2 fusions were transiently expressed in HEK293E cells cultured under standard conditions; 37 °C, 5% CO₂ in serum-free FreestyleTM 293 expression media (Gibco, #12338018). Polyethylenimine (transfection grade linear PEI MW 40kDa, PolySciences, #24765-1) and plasmid DNA were separately diluted in Optimem (Gibco, #11058-021), mixed in a 4:1 mass ratio and incubated for 15 min before mixing with FreeStyle 293 expression medium and added to 55–65% confluent HEK293E cells in 5-layer bottles (Corning, #353144). Complete protease inhibitor (Roche, #11873580001) was added to the conditioned medium and centrifuged (300 g, 10 min) before sterile filtering (Corning, #431098) and storage at 4 °C. Protein was harvested from supernatant and purified using a CaptureSelect human albumin affinity matrix (Thermo Fisher, cat#191297005) by elution with 2 M MgCl2 at pH 7.4. Concentration and buffer exchange into phosphate buffered saline (PBS) of the purified protein was performed using VivaSpin2 centrifugal concentrators (Sartorius, #VS0231). Subsequently, purified protein was snap frozen in liquid N2 and stored at -140 °C.
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