title: Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.
abstract: The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Gülin
authors.initials: G
authors.lastname: Güler-Gane
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Sara
authors.initials: S
authors.lastname: Kidd
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Sudharsan
authors.initials: S
authors.lastname: Sridharan
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Tristan J
authors.initials: TJ
authors.lastname: Vaughan
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Trevor C I
authors.initials: TC
authors.lastname: Wilkinson
authors.affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune Ltd., Granta Park, Cambridge, United Kingdom.
authors.forename: Natalie J
authors.identifier: 0000-0001-9916-0255
authors.initials: NJ
authors.lastname: Tigue
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D001692:Biological Transport; D017077:Culture Media, Conditioned / Q000737:chemistry; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D017077:Culture Media, Conditioned; D016898:Interferon-alpha; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase