{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:59712"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:59712","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Pflanzenzellen als Produktionssystem zur Gewinnung von Interleukin-4 Doppelmutein (IL-4 DM)","abstract":"The present study has emerged from the Bayer-FhG joint project on \"A feasibility study for recombinant protein production in plants\". Its objective was to analyse and evaluate the extent to which plant expression systems would be able to produce pharmaceutical proteins; the test protein chosen for this feasibility study was IL-4 DM (double mutein). The protein was expressed in tobacco plants and in tobacco suspension cells of the cultivar BY-2, making it possible to compare two fundamentally different plant production platforms. In order to increase the level of protein expression, the IL-4 DM gene sequence was optimised for plant cells; by drawing on PCR technology, synthetic versions of the gene were generated. The analysis of various gene constructs in transient transformations showed, however, that codon optimisation had no positive effect on the expression of IL-4 DM in tobacco. The accumulation of IL-4 DM was scrutinised in various cell compartments in both production systems. Comparing apoplasmatic localisation of the protein and ER-retardation of the protein, it was found that highest amounts of accumulation were given in the former case. Tobacco plants were stably transformed, and it was proven that apoplasmatically localised IL-4 DM was in fact expressed in transgenics of the T0 and the T1 generation; the protein yields reached up to 5 µg per gram leaf material. In transgenic BY-2 suspension cells, IL-4 DM was secreted highly efficiently and could be harvested from the culture supernatant directly. It was possible to increase the level of protein production by the factor 5 to 10, resulting in 1.5 to 3.8 µg per ml culture medium, by means of an overtransformation of transgenic BY-2 cell lines with IL-4 DM constructs carrying alternative IL-4 DM gene sequences and selection markers. Starting off from established purification protocols for bacterially expressed IL-4 DM protein, new strategies for the purification of the protein were developed for the plant systems at hand. The individual stages of purification were such that they utilised the physico-chemical properties of IL4-DM and that they made it possible to accumulate functional IL-4 DM that had been obtained both from leaf extract and from BY-2 culture supernatant. The functionality of IL-4 DM produced in plants was verified by means of in vitro binding to human cell lines overexpressing IL-4 receptor. Evidence was produced to show that the signal peptide was correctly cleaved from the IL-4 DM protein in the BY-2 system. In contrast, it transpired that 66% of the recombinant IL-4 DM from tobacco leaves included the last amino acid of the signal peptide. IL-4 DM is produced in plant cells in a glycosylated form. However, the patterns of glycosylation of the protein deviated marginally from the patterns of glycosylation typically found in mammalian cells, major reasons for such deviations being the presence of plant-specific Alpha-1,3 linked fucose and ß-1,2 linked xylose. Furthermore, it was shown that there are differences in the patterns of glycosylation of IL-4 DM between the BY-2 system on the one hand and tobacco leaves on the other. The results of biochemical tests gave evidence of the high quality of IL-4 DM produced in plants and thus proved the principal suitability of plant cells for the production of functional IL-4 DM.","abstract_html":"The present study has emerged from the Bayer-FhG joint project on "A feasibility study for recombinant protein production in plants". Its objective was to analyse and evaluate the extent to which plant expression systems would be able to produce pharmaceutical proteins; the test protein chosen for this feasibility study was IL-4 DM (double mutein). The protein was expressed in tobacco plants and in tobacco suspension cells of the cultivar BY-2, making it possible to compare two fundamentally different plant production platforms. In order to increase the level of protein expression, the IL-4 DM gene sequence was optimised for plant cells; by drawing on PCR technology, synthetic versions of the gene were generated. The analysis of various gene constructs in transient transformations showed, however, that codon optimisation had no positive effect on the expression of IL-4 DM in tobacco. The accumulation of IL-4 DM was scrutinised in various cell compartments in both production systems. Comparing apoplasmatic localisation of the protein and ER-retardation of the protein, it was found that highest amounts of accumulation were given in the former case. Tobacco plants were stably transformed, and it was proven that apoplasmatically localised IL-4 DM was in fact expressed in transgenics of the T0 and the T1 generation; the protein yields reached up to 5 µg per gram leaf material. In transgenic BY-2 suspension cells, IL-4 DM was secreted highly efficiently and could be harvested from the culture supernatant directly. It was possible to increase the level of protein production by the factor 5 to 10, resulting in 1.5 to 3.8 µg per ml culture medium, by means of an overtransformation of transgenic BY-2 cell lines with IL-4 DM constructs carrying alternative IL-4 DM gene sequences and selection markers. Starting off from established purification protocols for bacterially expressed IL-4 DM protein, new strategies for the purification of the protein were developed for the plant systems at hand. The individual stages of purification were such that they utilised the physico-chemical properties of IL4-DM and that they made it possible to accumulate functional IL-4 DM that had been obtained both from leaf extract and from BY-2 culture supernatant. The functionality of IL-4 DM produced in plants was verified by means of in vitro binding to human cell lines overexpressing IL-4 receptor. Evidence was produced to show that the signal peptide was correctly cleaved from the IL-4 DM protein in the BY-2 system. In contrast, it transpired that 66% of the recombinant IL-4 DM from tobacco leaves included the last amino acid of the signal peptide. IL-4 DM is produced in plant cells in a glycosylated form. However, the patterns of glycosylation of the protein deviated marginally from the patterns of glycosylation typically found in mammalian cells, major reasons for such deviations being the presence of plant-specific Alpha-1,3 linked fucose and ß-1,2 linked xylose. Furthermore, it was shown that there are differences in the patterns of glycosylation of IL-4 DM between the BY-2 system on the one hand and tobacco leaves on the other. The results of biochemical tests gave evidence of the high quality of IL-4 DM produced in plants and thus proved the principal suitability of plant cells for the production of functional IL-4 DM.","abstract_has_math":false,"creators":["Raven, Nicole"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fischer, Rainer"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2004,"date_issued":"2004","date_published":"2004","updated_at":"2026-07-30T19:42:48Z","subjects":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie","pflanzliche Expressionssysteme","Tabak","BY-2","rekombinante pharmazeutische Proteine","molecular farming"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208168%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208168%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208168%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/59712","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fischer, Rainer"]},{"key":"dc:creator","label":"Author","values":["Raven, Nicole"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2004"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-9918","info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-208168"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie","pflanzliche Expressionssysteme","Tabak","BY-2","rekombinante pharmazeutische Proteine","molecular farming"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/59712","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208168%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The present study has emerged from the Bayer-FhG joint project on \"A feasibility study for recombinant protein production in plants\". Its objective was to analyse and evaluate the extent to which plant expression systems would be able to produce pharmaceutical proteins; the test protein chosen for this feasibility study was IL-4 DM (double mutein). The protein was expressed in tobacco plants and in tobacco suspension cells of the cultivar BY-2, making it possible to compare two fundamentally different plant production platforms. In order to increase the level of protein expression, the IL-4 DM gene sequence was optimised for plant cells; by drawing on PCR technology, synthetic versions of the gene were generated. The analysis of various gene constructs in transient transformations showed, however, that codon optimisation had no positive effect on the expression of IL-4 DM in tobacco. The accumulation of IL-4 DM was scrutinised in various cell compartments in both production systems. Comparing apoplasmatic localisation of the protein and ER-retardation of the protein, it was found that highest amounts of accumulation were given in the former case. Tobacco plants were stably transformed, and it was proven that apoplasmatically localised IL-4 DM was in fact expressed in transgenics of the T0 and the T1 generation; the protein yields reached up to 5 µg per gram leaf material. In transgenic BY-2 suspension cells, IL-4 DM was secreted highly efficiently and could be harvested from the culture supernatant directly. It was possible to increase the level of protein production by the factor 5 to 10, resulting in 1.5 to 3.8 µg per ml culture medium, by means of an overtransformation of transgenic BY-2 cell lines with IL-4 DM constructs carrying alternative IL-4 DM gene sequences and selection markers. Starting off from established purification protocols for bacterially expressed IL-4 DM protein, new strategies for the purification of the protein were developed for the plant systems at hand. The individual stages of purification were such that they utilised the physico-chemical properties of IL4-DM and that they made it possible to accumulate functional IL-4 DM that had been obtained both from leaf extract and from BY-2 culture supernatant. The functionality of IL-4 DM produced in plants was verified by means of in vitro binding to human cell lines overexpressing IL-4 receptor. Evidence was produced to show that the signal peptide was correctly cleaved from the IL-4 DM protein in the BY-2 system. In contrast, it transpired that 66% of the recombinant IL-4 DM from tobacco leaves included the last amino acid of the signal peptide. IL-4 DM is produced in plant cells in a glycosylated form. However, the patterns of glycosylation of the protein deviated marginally from the patterns of glycosylation typically found in mammalian cells, major reasons for such deviations being the presence of plant-specific Alpha-1,3 linked fucose and ß-1,2 linked xylose. Furthermore, it was shown that there are differences in the patterns of glycosylation of IL-4 DM between the BY-2 system on the one hand and tobacco leaves on the other. The results of biochemical tests gave evidence of the high quality of IL-4 DM produced in plants and thus proved the principal suitability of plant cells for the production of functional IL-4 DM."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University IV, 185 S. : Ill., graph. Darst. (2004). doi:10.18154/RWTH-CONV-208168 = Aachen, Techn. Hochsch., Diss., 2004"]},{"key":"dc:title","label":"Title","values":["Pflanzenzellen als Produktionssystem zur Gewinnung von Interleukin-4 Doppelmutein (IL-4 DM)"]}]}],"canonical_facts":{"dc:contributor":["Fischer, Rainer"],"dc:coverage":["DE"],"dc:creator":["Raven, Nicole"],"dc:date":["2004"],"dc:description":["The present study has emerged from the Bayer-FhG joint project on \"A feasibility study for recombinant protein production in plants\". Its objective was to analyse and evaluate the extent to which plant expression systems would be able to produce pharmaceutical proteins; the test protein chosen for this feasibility study was IL-4 DM (double mutein). The protein was expressed in tobacco plants and in tobacco suspension cells of the cultivar BY-2, making it possible to compare two fundamentally different plant production platforms. In order to increase the level of protein expression, the IL-4 DM gene sequence was optimised for plant cells; by drawing on PCR technology, synthetic versions of the gene were generated. The analysis of various gene constructs in transient transformations showed, however, that codon optimisation had no positive effect on the expression of IL-4 DM in tobacco. The accumulation of IL-4 DM was scrutinised in various cell compartments in both production systems. Comparing apoplasmatic localisation of the protein and ER-retardation of the protein, it was found that highest amounts of accumulation were given in the former case. Tobacco plants were stably transformed, and it was proven that apoplasmatically localised IL-4 DM was in fact expressed in transgenics of the T0 and the T1 generation; the protein yields reached up to 5 µg per gram leaf material. In transgenic BY-2 suspension cells, IL-4 DM was secreted highly efficiently and could be harvested from the culture supernatant directly. It was possible to increase the level of protein production by the factor 5 to 10, resulting in 1.5 to 3.8 µg per ml culture medium, by means of an overtransformation of transgenic BY-2 cell lines with IL-4 DM constructs carrying alternative IL-4 DM gene sequences and selection markers. Starting off from established purification protocols for bacterially expressed IL-4 DM protein, new strategies for the purification of the protein were developed for the plant systems at hand. The individual stages of purification were such that they utilised the physico-chemical properties of IL4-DM and that they made it possible to accumulate functional IL-4 DM that had been obtained both from leaf extract and from BY-2 culture supernatant. The functionality of IL-4 DM produced in plants was verified by means of in vitro binding to human cell lines overexpressing IL-4 receptor. Evidence was produced to show that the signal peptide was correctly cleaved from the IL-4 DM protein in the BY-2 system. In contrast, it transpired that 66% of the recombinant IL-4 DM from tobacco leaves included the last amino acid of the signal peptide. IL-4 DM is produced in plant cells in a glycosylated form. However, the patterns of glycosylation of the protein deviated marginally from the patterns of glycosylation typically found in mammalian cells, major reasons for such deviations being the presence of plant-specific Alpha-1,3 linked fucose and ß-1,2 linked xylose. Furthermore, it was shown that there are differences in the patterns of glycosylation of IL-4 DM between the BY-2 system on the one hand and tobacco leaves on the other. The results of biochemical tests gave evidence of the high quality of IL-4 DM produced in plants and thus proved the principal suitability of plant cells for the production of functional IL-4 DM."],"dc:identifier":["https://publications.rwth-aachen.de/record/59712","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-208168%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-9918","info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-208168"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University IV, 185 S. : Ill., graph. Darst. (2004). doi:10.18154/RWTH-CONV-208168 = Aachen, Techn. Hochsch., Diss., 2004"],"dc:subject":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie","pflanzliche Expressionssysteme","Tabak","BY-2","rekombinante pharmazeutische Proteine","molecular farming"],"dc:title":["Pflanzenzellen als Produktionssystem zur Gewinnung von Interleukin-4 Doppelmutein (IL-4 DM)"],"dc:type":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]},"updated_at":"2026-07-30T19:42:48Z"}