title: Lentiviral expression system for the purification of secreted proteins from human cell cultures.
abstract: Recombinant proteins of therapeutic use are ideally produced in human cells to ensure appropriate co- and post-translational modifications. However, purification of secreted proteins from the culture media is impeded by low expression from transfected cell lines and the presence of serum proteins. Here we describe a simple and cost-effective approach based on lentiviral vector-mediated gene delivery and expression of a secreted His-tagged protein from human embryonic kidney 293 T cells and direct affinity chromatography purification from the cell culture media.
  
  
  
  Using a protein-based HIV entry inhibitor, soluble CD4 (sCD4), we demonstrated that 293 T cells transduced with a lentiviral vector mediated over 10-fold higher secretion of sCD4 in comparison to 293 T cells transfected with the corresponding plasmid. Secretion of sCD4 increased with the dose of the lentiviral vector up to a multiplicity of infection of 50. Exchanging the native signal peptide of sCD4 with the signal peptide of human alpha-1 antitrypsin increased expression by 50 %. There was no difference in expression from a monocistronic or bicistronic lentiviral vector. Reduction of the serum concentration in the culture media had no significant effect on the secretion of sCD4. Small-scale purification from 50 ml of culture media with reduced serum content yielded up to 1 mg of pure sCD4. Purified sCD4 bound to recombinant HIV envelope glycoprotein 120 (Env gp120) and inhibited HIV entry at concentrations comparable to published results.
  
  
  
  The procedure outlined in this study can be performed without the need for specialized reagents or equipment and could easily be adapted by any laboratory. Furthermore, the method could be used to produce sCD4 fusion proteins or other His-tagged proteins.
authors: Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Canada.
authors: Alexander
authors: A
authors: Falkenhagen
authors: Department of Pediatrics, The Hospital for Sick Children, Toronto, Canada.
authors: Sabah
authors: S
authors: Asad
authors: Department of Pediatrics, The Hospital for Sick Children, Toronto, Canada.
authors: Stanley E
authors: SE
authors: Read
authors: Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Canada. sadhna.joshi.sukhwal@utoronto.ca.|Department of Molecular Genetics, University of Toronto, Toronto, Canada. sadhna.joshi.sukhwal@utoronto.ca.
authors: Sadhna
authors: 0000-0001-5777-0493
authors: S
authors: Joshi
mesh_terms: D002846:Chromatography, Affinity; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006639:Histidine / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D006639:Histidine
keywords: His-tag; Human cells; Lentiviral vector; Purification; Secreted proteins; Soluble CD4