High-throughput measurements of protein domain functions using magnetic separation

High-throughput screening of protein domains enables systematic discovery of protein sequences that encode specific cellular functions. Fluorescence-activated cell sorting (FACS)-based assays have been the standard readout for such screens but remain time- and resource-intensive, imposing practical limits on library size and coverage. Here, we describe a scalable magnetic separation-based workflow that provides a simple and quantitative alternative to FACS for screening large protein libraries. We engineered a modular synthetic surface marker, consisting of an IgG1 Fc-PDGFRß transmembrane fusion, that allows cells to be magnetically separated based on surface reporter expression using Protein G-coated magnetic beads. This approach involves pooled library cloning, lentiviral delivery, magnetic separation, and sequencing-based quantification, enabling reproducible screening of over 200,000 protein domain variants. We have applied the method to transcriptional and RNA-regulatory effector domain screens, identifying new functional domains, and to the selection of improved transmembrane domains for efficient protein surface display. The entire workflow, from library design to data analysis, can be completed in three to four weeks and requires only basic cell culture, molecular cloning, and computational skills. This accessible, high-throughput protocol enables identification of functional protein domains across different phenotypes and provides an alternative screening readout for researchers without access to FACS facilities. Overall design: High-throughput protein domain screening in K562 cells, using magnetic separation to produce Bound and Unbound fractions each containing cells expressing different protein library members. Enrichment scores are calculated by the ratio of library member counts in each fraction. Libraries were designed as systematic mutations of the 'flexi-NR box' motif, LxxLL, and fused directly to the inducible DNA-binding domain rTetR. After recruitment to synthetic reporter genes under the control of either a minimal or a constitutively active promoter, protein domains were assessed for transcriptional activation or repression capacity.