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.
Highlights
The ability to recombinantly express and purify a protein of interest of sufficient quantity and quality is critical for many areas of biological science, not least in the production of biopharmaceuticals and recombinant antigens for therapeutic antibody generation
We identify amino acids that negatively influence the expression of secreted alkaline phosphatase (SEAP) when inserted at the +1 position and by investigating the expression of proteins with unfavourable native +1 amino acids we demonstrate that refractory expression can be restored by replacing the signal peptide or by placing small neutral amino acids at the +1/+2 positions
To investigate the effect of changing the signal peptide of SEAP to that of a non-native signal peptide, we first chose to investigate the commonly used human CD33 signal peptide that has been shown to improve the secretion of proteins such as IL-15 [14]
Summary
The ability to recombinantly express and purify a protein of interest of sufficient quantity and quality is critical for many areas of biological science, not least in the production of biopharmaceuticals and recombinant antigens for therapeutic antibody generation In the latter case, the target antigen needs to be available in a form that most closely resembles the native protein. The target antigen needs to be available in a form that most closely resembles the native protein Prokaryotic expression hosts such as E. coli provide a convenient and cost-effective means for producing recombinant proteins, the proteins obtained via these systems are not glycosylated and there is a risk that they will not be folded properly [1]. In this study we sought to identify a universal strategy to improve the expression of secreted recombinant proteins in HEK293 cells, by investigating the role of N-terminal signal peptides
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