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High-throughput measurements of protein domain functions using magnetic separation

ABSTRACT: 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-PDGFRβ 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE317498 | GEO | 2026/01/28

REPOSITORIES: GEO

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			Action	DRS
		Other
	GSE317498_flexiNR_ActD2_combo.csv.gz	Csv
	GSE317498_flexiNR_ReprD5_combo.csv.gz	Csv

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Project description:Background Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed. Results We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach. Conclusions The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning. Keywords: Xenoarray - a barcode-type microarray-based functional screening.

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High-throughput measurements of protein domain functions using magnetic separation

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