An improved workflow for rapid, large-scale protein production in HEK293 cells via antibiotic enrichment after lentiviral transduction

Lentiviral transduction of HEK293-derived expression cells provides a robust and scalable approach for large-scale protein production for structural and biochemical studies. Building on our previously reported platform, we introduce an improved workflow that decouples cell enrichment from target protein expression by enabling constitutive antibiotic selection of transduced cells prior to induction. The key advance is the use of orthogonal antibiotic-resistance cassettes to stringently enrich transduced cells, eliminate non-transduced cells, improve population homogeneity, and enable multi-vector co-selection for heteromeric assemblies and complexes. We provide two complementary transfer-vector suites. pHR-AB-CMV-TetO2 delivers maximal expression and supports inducible control in TetR-expressing lines while driving strong constitutive expression in non-TetR lines. pHR-AIO-AB ("all-in-one") encodes the transactivator, resistance marker, and gene of interest on a single construct to enable tightly controlled doxycycline-inducible expression in standard HEK293 lines, and is readily adaptable to other mammalian cell types. Both suites are available with puromycin, blasticidin, hygromycin, or zeocin markers, enabling straightforward co-infection and orthogonal multi-antibiotic selection of stable populations expressing multiple transgenes. They are well suited to demanding targets such as membrane proteins and multi-subunit assemblies. The protocol details the step-by-step generation of highly enriched, inducible HEK293 populations within 3-4 weeks.