Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids

<div><p>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.</p></div

Western blot quantification of secreted SEAP levels using different signal peptides.

<p>SEAP-encoding constructs were transiently expressed in HEK293 cells and the level of secreted SEAP in the conditioned media was visualised by Western blotting. Full-size Western blots are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155340#pone.0155340.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155340#pone.0155340.s005" target="_blank">S5 Fig</a>. The functional activity of SEAP within the conditioned media was measured using a chemiluminescent substrate (Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (Clontech, Takara)). The amount of SEAP was calculated based on linear regression analysis using a standard curve, and the percentage change compared to the control sample (Native-SEAP), was determined. Statistical analyses for each of the duplicate experiments were performed using a one-way ANOVA (Dunnett’s correction for multiple comparisons; **** = P ≤ 0.0001). Data for each sample from the duplicate experiments is shown side-by-side to highlight inter-experimental variability. Error bars represent the standard error of the mean from six replicate assay wells.</p

Western blot quantification of secreted SEAP levels using different +1 residues.

<p>Secrecon-SEAP-encoding constructs were transiently expressed in HEK293 cells and the level of secreted SEAP in the conditioned media was visualised by Western blotting. Panels A and B represent 2 separate Western blots representing the data from the 18 different samples. Full-size Western blots are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155340#pone.0155340.s006" target="_blank">S6 Fig</a>. The functional activity of SEAP within the conditioned media was measured using a chemiluminescent substrate (Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (Clontech, Takara)). The amount of SEAP was calculated based on linear regression analysis using a standard curve, and the percentage change compared to the control sample (Native-SEAP), was determined. Statistical analyses for each of the duplicate experiments were performed using a one-way ANOVA (Dunnett’s correction for multiple comparisons; **** = P ≤ 0.0001). Data for each sample from the duplicate experiments is shown side-by-side to highlight inter-experimental variability. Error bars represent the standard error of the mean from six replicate assay wells.</p

Western blot quantification of secreted SEAP levels using CD33 signal peptides.

<p>SEAP-encoding constructs were transiently expressed in HEK293 cells and the level of secreted SEAP in the conditioned media was visualised by Western blotting Full-size Western blots are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155340#pone.0155340.s002" target="_blank">S2 Fig</a>. The functional activity of SEAP within the conditioned media was measured using a chemiluminescent substrate (Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (Clontech, Takara)). The amount of SEAP was calculated based on linear regression analysis using a standard curve, and the percentage change compared to the control sample (Native-SEAP), was determined. Statistical analyses for each of the duplicate experiments were performed using a one-way ANOVA (Dunnett’s correction for multiple comparisons; **** = P ≤ 0.0001). Data for each sample from the duplicate experiments is shown side-by-side to highlight inter-experimental variability. Error bars represent the standard error of the mean from six replicate assay wells.</p

Summary of the signal peptides used in this study.

<p>Summary of the signal peptides used in this study.</p

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and <i>in vivo</i>, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development