Extended Data Fig. 3: Expression, purification, and Fab complex formation of MmFLVCR2.
From: Structural and molecular basis of choline uptake into the brain by FLVCR2
a, Fluorescent size exclusion chromatography elution profiles of six FLVCR2 orthologs fused to green fluorescent protein (GFP). Orthologs screened are from Homo sapiens (HsFLVCR2; NCBI – NP_060261.2), Mus musculus (MmFLVCR2; NCBI – NP_663422.1), Eptesicus fuscus (EfFLVCR2; NCBI – XP_027997147.2), Canis lupus dingo (CldFLVCR2; NCBI – XP_025299773.1), Bos taurus (BtFLVCR2; NCBI – NP_001179072.1), and Danio rerio (DrFLVCR2; NCBI – XP_693589.3). b, Representative SDS-PAGE gel of MmFLVCR2 purified in DDM/CHS. c, EC50 evaluation of select purified Fabs binding to MmFLVCR2 incorporated into MSP1D1 nanodisc. Data points represent the mean of two data points; approximate EC50 values are provided. d, Normalised high-performance liquid chromatography elution profiles of MmFLVCR2 reconstituted into MSP1D1 nanodisc alone (black) and in complex with Fab FLV23 (blue) and Fab FLV9 (orange). Numbers indicate the retention time (mins) of each peak. e, Mass photometry analysis of nanodisc-reconstituted MmFLVCR2 purified via size exclusion chromatography in the presence (orange) and absence (purple) of Fab FLV23. The peaks differ by 51 kDa which corresponds to the molecular weight of Fab FLV23. Note that negative masses are artefacts caused by non-protein unbinding events. f, Single-point phage ELISA for binding of MmFLVCR2-specific Fab-phage in the presence (orange) and absence (blue) of 1 μM purified Fab FLV32, which was used as an epitope masking reagent. Note that FLV32 was confirmed to have the same binding epitope as FLV23 via preliminary cryo-EM experiments (data not shown). Only Fab FLV9 produced the same level of ELISA signal in the presence and absence of the Fab FLV32.
