Escherichia coli bl21 star de3

The affibody constructs were expressed at 37 °C in shake flask cultures of Escherichia coli BL21 Star (DE3) (New England Biolabs) in tryptic soy broth containing 100 µg/mL ampicillin. When OD₆₀₀ was between 0.6 and 1, protein expression was induced by addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK). Protein production was carried out for 3 h, after which the cells were harvested by centrifugation and lysed by sonication. The supernatants were clarified by centrifugation and filtration through a 0.45 µm Acrodisc syringe filter (Pall, Port Washington, NY, USA). The recombinantly expressed affibody constructs were purified by affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) with immobilized human serum albumin (HSA) using an ÄKTA system (GE Healthcare), essentially as previously described [44 (link)] including elution with 500 mM acetic acid, pH 2.6. The fractions containing affibody constructs were pooled and lyophilized.
Z_HER2:V2 was produced and purified as previously described [45 (link)].

The affibody/ABD fusion proteins were expressed in Escherichia coli BL21 Star (DE3) (New England Biolabs), grown in tryptic soy broth supplemented with 5 g/L yeast extract and 100 μg/mL ampicillin. The cells were grown at 37 °C in shake flasks. Expression was induced by adding 1 mM of isopropyl β-d-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK) when the OD₆₀₀ was between 0.6 and 1.0. The cells were cultured for another 3 h at 37 °C. The cell suspension was centrifuged and the cytoplasmic fraction was released by sonication. The cell lysate was clarified by passage through a 0.45 µm Acrodisc syringe filter (Pall, Port Washington, NY, USA). Human serum albumin-based affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) was performed to isolate the ABD fused affibody constructs. The purification was carried out on an ÄKTA system (GE Healthcare Life Sciences, Uppsala, Sweden), essentially as previously described [20 (link)]. Elution was carried out with 500 mM acetic acid (pH = 2.6). The fractions containing affibody fusion proteins were pooled and lyophilized.

The affibody constructs were expressed at 37 °C in shake flask cultures of Escherichia coli BL21 Star (DE3) (New England Biolabs). When OD₆₀₀ was between 0.6 and 1, protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (Appolo Scientific, Stockport, UK). Protein production was carried out for 3 h, after which the cells were harvested by centrifugation and lysed by sonication. The supernatants were clarified by centrifugation and filtration through a 0.45 μm Acrodisc syringe filter (Pall, Port Washington, NY, USA). The recombinantly expressed affibody constructs were purified by affinity chromatography on a HiTrap NHS sepharose column (GE Healthcare, Uppsala, Sweden) with immobilized human serum albumin (HSA) using an ÄKTA system (GE Healthcare), essentially as previously described [14 (link)] including elution with 50 mM acetic acid. The fractions containing affibody constructs were pooled and lyophilized.