In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger
The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a hos...
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University of Zagreb Faculty of Food Technology and Biotechnology
2003-01-01
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| Series: | Food Technology and Biotechnology |
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| Online Access: | http://hrcak.srce.hr/file/185064 |
| _version_ | 1797426355058507776 |
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| author | Monika Svetina Vladka Gaberc-Porekar Viktor Menart Radovan Komel Nada Kraševec |
| author_facet | Monika Svetina Vladka Gaberc-Porekar Viktor Menart Radovan Komel Nada Kraševec |
| author_sort | Monika Svetina |
| collection | DOAJ |
| description | The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis. |
| first_indexed | 2024-03-09T08:29:02Z |
| format | Article |
| id | doaj.art-a418de7300d04869a8dd208072c90916 |
| institution | Directory Open Access Journal |
| issn | 1330-9862 1334-2606 |
| language | English |
| last_indexed | 2024-03-09T08:29:02Z |
| publishDate | 2003-01-01 |
| publisher | University of Zagreb Faculty of Food Technology and Biotechnology |
| record_format | Article |
| series | Food Technology and Biotechnology |
| spelling | doaj.art-a418de7300d04869a8dd208072c909162023-12-02T20:38:56ZengUniversity of Zagreb Faculty of Food Technology and BiotechnologyFood Technology and Biotechnology1330-98621334-26062003-01-01414345351In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus nigerMonika Svetina0Vladka Gaberc-Porekar1Viktor Menart2Radovan Komel3Nada Kraševec4National Institute of Chemistry, POB 660, SI-1001 Ljubljana, SloveniaNational Institute of Chemistry, POB 660, SI-1001 Ljubljana, SloveniaNational Institute of Chemistry, POB 660, SI-1001 Ljubljana, SloveniaNational Institute of Chemistry, POB 660, SI-1001 Ljubljana, SloveniaNational Institute of Chemistry, POB 660, SI-1001 Ljubljana, SloveniaThe most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis.http://hrcak.srce.hr/file/185064filamentous fungiAspergillus nigerheterologous protein secretionhuman tumor necrosis factor α (TNFα) KEX2 cleavage siteenterokinase cleavage siteHis-tag |
| spellingShingle | Monika Svetina Vladka Gaberc-Porekar Viktor Menart Radovan Komel Nada Kraševec In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger Food Technology and Biotechnology filamentous fungi Aspergillus niger heterologous protein secretion human tumor necrosis factor α (TNFα) KEX2 cleavage site enterokinase cleavage site His-tag |
| title | In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger |
| title_full | In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger |
| title_fullStr | In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger |
| title_full_unstemmed | In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger |
| title_short | In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger |
| title_sort | in vivo and in vitro cleavage of glucoamylase tnfα fusion protein secreted from aspergillus niger |
| topic | filamentous fungi Aspergillus niger heterologous protein secretion human tumor necrosis factor α (TNFα) KEX2 cleavage site enterokinase cleavage site His-tag |
| url | http://hrcak.srce.hr/file/185064 |
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