The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector containing the hIgG1 Fc regions (hFc) at the C-terminus as described previously [67 (link)]. The expression vectors were transfected into HEK293F cells (Invitrogen), and the fusion proteins were purified by Protein A affinity chromatography as described previously [67 (link)].
For the expression of IgG1, genes encoding VH and VL were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgG1 was purified by Protein A affinity chromatography as described previously [68 (link)]. Then the eluate containing IgG1 was subjected to gel filtration chromatography. A total of 4 mg of IgG1 was injected at a flow rate of 1 mL/min and purified by gel filtration using a XK16/100 column packed with Superdex 200 pg at pH 7.4 (ÄKTA pure, GE Healthcare). The chromatogram was recorded at a UV absorbance of 280 nm. The fractions containing IgG1 were pooled by collection criteria and concentrated.
For the expression of IgG1, genes encoding VH and VL were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgG1 was purified by Protein A affinity chromatography as described previously [68 (link)]. Then the eluate containing IgG1 was subjected to gel filtration chromatography. A total of 4 mg of IgG1 was injected at a flow rate of 1 mL/min and purified by gel filtration using a XK16/100 column packed with Superdex 200 pg at pH 7.4 (ÄKTA pure, GE Healthcare). The chromatogram was recorded at a UV absorbance of 280 nm. The fractions containing IgG1 were pooled by collection criteria and concentrated.
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