Fab Expression and Purification for Structural Studies

The CR3022 Fab heavy (GenBank: DQ168569.1) and light (GenBank: DQ168570.1) chains were cloned into phCMV3. The plasmids were transiently co-transfected into Expi293F cells at a ratio of 2:1 (HC:LC) using ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The supernatant was collected at 7 days post-transfection. The Fab was purified with a CaptureSelect™ CH1-XL Pre-packed Column (Thermo Fisher Scientific) followed by size exclusion chromatography. For crystallization, a VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the constant and variable domains [25 (link)].

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Wu N.C., Yuan M., Bangaru S., Huang D., Zhu X., Lee C.C., Turner H.L., Peng L., Yang L., Nemazee D., Ward A.B, & Wilson I.A. (2020). A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody. bioRxiv.

Publication Preprint 2020

ExpiFectamine™ 293 Reagent CaptureSelect™ CH1-XL Pre-packed Column

Cells Cr3022 Crystallization Light Plasmids Region of elbow Size exclusion chromatography Transfection

Corresponding Organization : Scripps Research Institute

Other organizations : University of Illinois Urbana-Champaign

Top 5 similar protocols

Variable analysis

independent variables

Ratio of heavy chain (HC) to light chain (LC) plasmids (2:1)

dependent variables

Fab protein production and purification

control variables

Expi293F cells
ExpiFectamine™ 293 Reagent (Thermo Fisher Scientific)
CaptureSelect™ CH1-XL Pre-packed Column (Thermo Fisher Scientific)
Size exclusion chromatography

controls

VSRRLP variant of the elbow region was used to reduce the conformational flexibility between the constant and variable domains

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