High-Level Production of Recombinant Eukaryotic Proteins from Mammalian Cells Using Lentivirus

Methods Mol Biol. 2021:2305:83-104. doi: 10.1007/978-1-0716-1406-8_4.


Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO2, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviral titer, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production. The typical lead-time for a full production run is ~3-4 weeks, with an anticipated yield of up to tens of milligrams of protein per liter of expression medium.

Keywords: Flow cytometry; HEK293 cells; Lentivirus; Membrane proteins; Recombinant protein production; Stable cell lines; Structural biology.

MeSH terms

  • Animals
  • Biotechnology / methods*
  • Cell Culture Techniques / methods*
  • Cell Line
  • Genetic Vectors
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics
  • Lentivirus / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Plasmids / genetics
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics
  • Transduction, Genetic / methods
  • Transfection / methods


  • Membrane Proteins
  • Recombinant Proteins