Fab Purification for Crystallization

H7.5 Fab for crystallization was prepared as previously described [30 (link)]. In brief, the heavy and light chains of H7.5 were cloned independently into the phCMV3 vector and fused with an N-terminal IgK secretion signal. A His₆ tag was added to the C-terminus of the Fab heavy chain. Recombinant cDNAs encoding the Fab heavy and light chains were purified and cotransfected into 293F cells by 293fectin (Invitrogen). After 6–7 days of expression at 37 °C, the Fabs were purified from the supernatant by Ni-NTA Superflow (Qiagen) and monoS chromatography (GE Healthcare).

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Turner H.L., Pallesen J., Lang S., Bangaru S., Urata S., Li S., Cottrell C.A., Bowman C.A., Crowe JE J.r., Wilson I.A, & Ward A.B. (2019). Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains. PLoS Biology, 17(2), e3000139.

Publication 2019

293fectin Ni-NTA Superflow monoS chromatography

Cdnas Cells Chromatography Crystallization His6 tag Light Monos Secretion Vector

Corresponding Organization : Vanderbilt University Medical Center

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Variable analysis

independent variables

Preparation of H7.5 Fab for crystallization

dependent variables

Purification of Fabs from the supernatant

control variables

Expression at 37 °C for 6-7 days
Purification by Ni-NTA Superflow (Qiagen) and monoS chromatography (GE Healthcare)

controls

No positive or negative controls were explicitly mentioned.

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