Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids
Publication Date
May 19, 2016
Journal
PLOS ONE
Authors
Gülin Güler Gane, Sara Kidd, Sudharsan Sridharan, Tristan J. Vaughan, et al
Volume
11
Issue
5
Pages
e0155340
DOI
https://dx.plos.org/10.1371/journal.pone.0155340
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0155340
Web of Science
000376291100037
Scopus
84982167173
Mendeley
http://www.mendeley.com/research/overcoming-refractory-expression-secreted-recombinant-proteins-mammalian-cells-through-modification
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Mendeley | Further Information

{"title"=>"Overcoming the refractory expression of secreted recombinant proteins in mammalian cells through modification of the signal peptide and adjacent amino acids", "type"=>"journal", "authors"=>[{"first_name"=>"Gülin", "last_name"=>"Güler-Gane", "scopus_author_id"=>"55763792804"}, {"first_name"=>"Sara", "last_name"=>"Kidd", "scopus_author_id"=>"57190660235"}, {"first_name"=>"Sudharsan", "last_name"=>"Sridharan", "scopus_author_id"=>"7102172677"}, {"first_name"=>"Tristan J.", "last_name"=>"Vaughan", "scopus_author_id"=>"7103008915"}, {"first_name"=>"Trevor C.I.", "last_name"=>"Wilkinson", "scopus_author_id"=>"7202351263"}, {"first_name"=>"Natalie J.", "last_name"=>"Tigue", "scopus_author_id"=>"7801575041"}], "year"=>2016, "source"=>"PLoS ONE", "identifiers"=>{"doi"=>"10.1371/journal.pone.0155340", "issn"=>"19326203", "sgr"=>"84982167173", "pui"=>"611667896", "scopus"=>"2-s2.0-84982167173", "pmid"=>"27195765", "isbn"=>"1932-6203 (Electronic) 1932-6203 (Linking)"}, "id"=>"6f77393a-81b2-3e6c-a6d5-e6cc3a46aad9", "abstract"=>"The expression and subsequent purification of mammalian recombinant proteins is of criti-cal 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, how-ever, 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 pep-tides and residues immediately downstream, in influencing the level of secreted recombi-nant 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 het-erologous 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 spe-cific 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 pro-teins that have so far proved refractory to expression in HEK293 cells, to be produced in suf-ficient quantities to answer important biological questions.", "link"=>"http://www.mendeley.com/research/overcoming-refractory-expression-secreted-recombinant-proteins-mammalian-cells-through-modification", "reader_count"=>52, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>13, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>11, "Other"=>3, "Student > Bachelor"=>7, "Lecturer"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>3, "Researcher"=>13, "Student > Doctoral Student"=>5, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>11, "Other"=>3, "Student > Bachelor"=>7, "Lecturer"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Engineering"=>5, "Biochemistry, Genetics and Molecular Biology"=>20, "Agricultural and Biological Sciences"=>16, "Medicine and Dentistry"=>2, "Neuroscience"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>5}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>16}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>20}, "Unspecified"=>{"Unspecified"=>4}}, "reader_count_by_country"=>{"Denmark"=>1, "United Kingdom"=>1}, "group_count"=>1}

Scopus | Further Information

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