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Use of Endogenous Signal Sequences for Transient Production and Efficient BMC Biotechnology BioMed Central Research article Open Access Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells Andreas Schaaf†3, Stefanie Tintelnot†1, Armin Baur1,2, Ralf Reski1, Gilbert Gorr2 and Eva L Decker*1 Address: 1Department of Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, Germany, 2greenovation Biotech GmbH, Boetzinger Str. 29b, 79111 Freiburg, Germany and 3Department of Plant Biochemistry and Biotechnology, University of Münster, Hindenburgplatz 55, 48143 Münster, Germany Email: Andreas Schaaf - [email protected]; Stefanie Tintelnot - [email protected]; Armin Baur - [email protected]; Ralf Reski - [email protected]; Gilbert Gorr - [email protected]; Eva L Decker* - [email protected] * Corresponding author †Equal contributors Published: 07 November 2005 Received: 20 May 2005 Accepted: 07 November 2005 BMC Biotechnology 2005, 5:30 doi:10.1186/1472-6750-5-30 This article is available from: http://www.biomedcentral.com/1472-6750/5/30 © 2005 Schaaf et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. Background modification like erythropoietin or coagulation factor IX The product range for recombinant biopharmaceuticals [1,2]. Commercial-scale production of proteins (molecu- includes relatively simple proteins, for example insulin, lar farming) nowadays is mainly performed with bacteria and ends with candidates that require extensive secondary or mammalian cell-culture systems [1,3,4]. Bacterial pro- Page 1 of 11 (page number not for citation purposes) BMC Biotechnology 2005, 5:30 http://www.biomedcentral.com/1472-6750/5/30 duction systems are highly efficient and well established between plants and animals, they are not universally for the production of comparably simple proteins but interchangeable [18]. Additional features might contrib- they come to their limits if complex posttranslational ute to the efficiency of signal peptide recognition [19]. In processing is required. One major problem is the absence order to establish moss as a production system for recom- of protein glycosylation in prokaryotes. Mammalian cell binant proteins, we intended to enrich our toolboxes with lines, on the other hand, perform human-like posttransla- a set of endogenous signal sequences from the moss. The tional modifications and offer a high product quality but availability of Physcomitrella secretion signals gives us the hold risks of product contamination and high overall pro- opportunity to secrete even proteins that are located duction costs (e.g. [5]). within the cytosol in their host organism and thus lack a secretion signal. Plants have emerged as a safe and cost-effective alternative to the traditional systems (e.g. [6,7]). They synthesize pro- Here, we describe the comparison of two heterologous teins with correct assemblage and the posttranslational signal peptides from human origin, the vascular endothe- modifications of higher eukaryotes. In contrast to micro- lial growth factor (hVEGF) and the coagulation factor IX bial systems, glycosylated plant proteins carry complex- (hFIX), respectively, in the moss. Evaluation of a heterol- type glycans with the same core structure as human glyco- ogous signal and the signal peptide of Physcomitrella aspar- proteins. The glycosylation pattern of plant proteins, how- tic proteinase PpAP1 [20] revealed better secretion of ever, differs from that in humans concerning two residues recombinant hVEGF (rhVEGF) with the moss signal. linked to the core structure as well as terminal sugar moi- eties [2,8]. The adaptation of the plant glycan pattern to its In addition, the secretion efficiency of five putative signal human counterpart (humanisation) poses a challenge for peptides originating from recently isolated and identified the implementation of plant systems with regard to the Physcomitrella extracellular proteins xyloglucan endotrans- production of recombinant pharmaceutical proteins [9] glycosylase/hydrolase (PpXTH1), pectin methylesterase as differences in the glycan structure could substantially (PpPME1), fasciclin-like protein (PpFLP), carbonic anhy- alter protein characteristics and even act immunogenically drase-like protein (PpCALP), and lipid-transfer protein in patients [10-13]. (PpLTP) (Tintelnot et al., manuscript in preparation) was tested in comparison to the PpAP1 secretion signal. These The moss Physcomitrella patens is the only known plant in signal sequences were fused to hVEGF and amounts of which homologous recombination occurs in a frequency secreted rhVEGF were quantified by ELISA measurements. that allows its application as an engineering tool for tar- All of the investigated moss signal peptides showed at geted knock out and gene replacement. With this tech- least comparable or even higher secretion efficiency than nique a humanisation of the moss glycosylation pattern the human signals. Besides, we underline the functional- was performed recently [14,15]. Together with photoau- ity of a transient production technique, that allows the totrophic growth in bioreactors these advantages make fast production of recombinant proteins [21]. Within the Physcomitrella an ideal system for the production of plant- field of molecular farming these techniques are essential made pharmaceuticals [16]. for proof-of-concept studies prior to the establishment of production lines. In molecular farming approaches, recombinant proteins, which require correct posttranslational modifications, are Results often targeted to the secretory pathway. This, on the one Efficiency of human secretion signals in Physcomitrella hand, is due to the fact that enzymes responsible for vari- patens ous modifications, e. g. glycosylation and protein trim- In order to improve transient production and secretion of ming, are located within the membranes of endoplasmic pharmaceutically interesting proteins in the moss Phys- reticulum (ER) and Golgi apparatus. On the other hand, comitrella patens, we first tested the efficiency of two secretion of the protein of interest into the culture human signal peptides. These were derived from the medium separates it from the pool of intracellular pro- human blood clotting factor IX (hFIX) and the vascular teins and therefore drastically facilitates its purification endothelial growth factor (hVEGF). The secretion signals and reduces costs for downstream processing [1]. of both proteins, FSP and VSP, respectively, were tested with hVEGF as a reporter gene. Physcomitrella protoplasts A common way to achieve secretion of recombinant pro- were transiently transfected with the respective plasmids teins is the utilisation of the native signal peptide, if any is followed by quantification of extra- and intracellular con- present. The mechanism of protein secretion depends on centrations of the recombinant proteins by ELISA (Fig. 1). the recognition of an N-terminal signal peptide and is Testing the hFIX signal peptide FSP, the moss cells conserved among all eukaryotes [17]. Though the general secreted on average 18.17 ng/ml recombinant hVEGF hydrophobic character of secretion signals is conserved (rhVEGF) to the culture supernatant, whereas the hVEGF Page 2 of 11 (page number not for citation purposes) BMC Biotechnology 2005, 5:30 http://www.biomedcentral.com/1472-6750/5/30 situation. ASP-GST-transfected protoplasts contained 3.38 35 ng/ml rGST whereas those transfected with VSP-GST 30 reached a mean value of no more than 0.35 ng/ml. 25 For visualisation of recombinant GST on Western blots 20 (Fig. 2),
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