Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

Abstract

Background: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition.

Results: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides.

Conclusion: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems.

Figure 1

Comparison of heterologous signals from human origin. The secretion signals of human blood clotting factor IX (FSP) as well as human vascular endothelial growth factor (VSP) were fused to the coding sequence of vascular endothelial growth factor (hVEGF). Moss protoplasts were transiently transfected with the expression constructs and concentration of extracellular as well as intracellular rhVEGF was determined by ELISA. Mean values were taken from three transfections. Error bars indicate the absolute average deviation (AAD).

Figure 2

Concentrations of secreted recombinant GST. Physcomitrella protoplasts were transiently transfected with pVSP-GST and pASP-GST in eight independent transfections. After 5 days the concentrations of secreted and intracellularly-retained recombinant protein were measured by ELISA. Additionally, GST was affinity-purified from both medium and supernatant and detected by western blot. A: Culture medium of two transfections was pooled. Mean values of eight transfections are given. B: Protoplasts of eight transfections were pooled and GST was affinity-purified from the crude extracts. Values reflect the means of the two measurements. Control: mock-transfected protoplasts.

Figure 3

Intra- and extracellular concentrations of recombinant rhVEGF. Physcomitrella protoplasts were transiently transfected with pVSP-VEGF and pASP-VEGF in four (pVSP-VEGF and control) and seven (pASP-VEGF) independent transfections. After 5 days the concentrations of secreted and intracellularly retained recombinant protein were measured by ELISA. A: mean values of concentrations of extra- and intracellular rhVEGF measured by ELISA. B: Western blot with culture medium of transiently rhVEGF-producing cell. Control: untransfected moss protoplasts.

Figure 4

Intra- and extracellular distribution of recombinant protein. The relations between intra- and extracellular amounts of recombinant rhVEGF (A) and GST (B) are given. Values reflect the mean amounts of recombinant protein per transfection. ASP: plasmids with moss signal peptide; VSP: plasmids with human signal peptide; extra: amount of recombinant protein in culture medium; intra: intracellular amounts of recombinant protein.

Figure 5

Secretion efficiency of different moss signal peptides. Signal sequences of PpAP1, PpFLP, PpLTP, PpPME1, PpXTH1, PpCALP were fused to rhVEGF. Shown are the mean values of three independent experiments with three transfections each time (n = 9). Prior to calculation of averages, outliers were removed if the z-score was >1. RhVEGF concentration of the PpAP1-construct was set to 100%. Error bars indicate the absolute average deviation (AAD) of the three experiments. Control: untransfected Physcomitrella protoplasts.

Figure 6

Physcomitrella patens signal peptides. Amino acid sequences of signal peptides from PpAP1 (ASP), VEGF (VSP) and the extracellular proteins PpFLP, PpLTP, PpPME1, PpXTH1, PpCALP, and VEGF are shown as predicted by SignalP. Letters with yellow background represent hydrophobic amino acid residues (V, L, I, W, F, M), orange letters positive (K, R) and green letters small and neutral residues (A, C, G, N, P, S, T, V). The predicted cleavage sites are indicated as well as the positions adjacent to this site. In the right part of the figure probabilities for each signal peptide and the cleavage site given by the SignalP-HMM prediction as well as the length of the signal peptides are shown.