title: Automation of large scale transient protein expression in mammalian cells.
abstract: Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.
authors.affiliation: Division of Structural Biology (STRUBI), Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX37BN, UK.
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bishop
authors.forename: Jordan E
authors.initials: JE
authors.lastname: Clay
authors.forename: Weixian
authors.initials: W
authors.lastname: Lu
authors.forename: Margaret
authors.initials: M
authors.lastname: Jones
authors.forename: Susan
authors.initials: S
authors.lastname: Daenke
authors.forename: Christian
authors.initials: C
authors.lastname: Siebold
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: A Radu
authors.initials: AR
authors.lastname: Aricescu
mesh_terms: D057205:Automation, Laboratory / Q000295:instrumentation* / Q000379:methods; D018929:Cell Culture Techniques; D004270:DNA, Circular / Q000302:isolation & purification; D057809:HEK293 Cells; D053823:Hedgehog Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D006801:Humans; D010957:Plasmids / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection / Q000379:methods
chemical_list: D004270:DNA, Circular; D053823:Hedgehog Proteins; D011994:Recombinant Proteins; C465570:Shh protein, mouse