{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,4]],"date-time":"2025-11-04T09:44:47Z","timestamp":1762249487678},"reference-count":24,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2014,8,1]],"date-time":"2014-08-01T00:00:00Z","timestamp":1406851200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"},{"start":{"date-parts":[[2014,8,1]],"date-time":"2014-08-01T00:00:00Z","timestamp":1406851200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/www.springer.com\/tdm"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,8,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Production of recombinant protein\u00a0bio-insecticides on a\u00a0commercial scale can only be cost effective if host\u00a0strains with very high expression levels are available. A recombinant fusion protein containing an arthropod toxin, \u03c9-hexatoxin-Hv1a, (from funnel web spider Hadronyche versuta) linked to snowdrop lectin (Galanthus nivalis agglutinin; GNA) is an effective oral insecticide and candidate biopesticide. However, the fusion protein was vulnerable to proteolysis during production in the yeast Pichia pastoris. To prevent proteolysis, the Hv1a\/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a\/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. Removal of the Kex2 site resulted in increased levels of intact fusion protein expressed in wild-type P. pastoris strains, improving levels of intact recombinant protein recoverable. Incorporation of a C-terminal (His)6 tag enabled single step purification of the fusion protein. These modifications did not affect the insecticidal activity of the recombinant toxin towards lepidopteran larvae. Introduction of multiple expression cassettes increased the amount of secreted recombinant fusion protein in a laboratory scale fermentation by almost tenfold on a per litre of culture basis. Simple modifications in the expression construct can be advantageous for the generation of high expressing P. pastoris strains for production of a recombinant protein, without altering its functional properties.<\/jats:p>","DOI":"10.1007\/s10295-014-1466-8","type":"journal-article","created":{"date-parts":[[2014,6,4]],"date-time":"2014-06-04T22:10:41Z","timestamp":1401919841000},"page":"1237-1247","update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Optimising expression of the recombinant fusion protein biopesticide \u03c9-hexatoxin-Hv1a\/GNA in<i>Pichia pastoris:<\/i>sequence modifications and a simple method for the generation of multi-copy strains"],"prefix":"10.1093","volume":"41","author":[{"given":"Prashant","family":"Pyati","sequence":"first","affiliation":[{"name":"grid.8250.f 0000000087000572 School of Biological and Biomedical Sciences Durham University South Road DH1 3LE Durham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elaine","family":"Fitches","sequence":"additional","affiliation":[{"name":"grid.13689.35 0000000404261697 The Food and Environment Research Agency Sand Hutton YO41 1LZ York UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John A","family":"Gatehouse","sequence":"additional","affiliation":[{"name":"grid.8250.f 0000000087000572 School of Biological and Biomedical Sciences Durham University South Road DH1 3LE Durham UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2014,8,1]]},"reference":[{"key":"2021033103281719200_CR1","doi-asserted-by":"publisher","first-page":"1281","DOI":"10.1023\/A:1025007901322","article-title":"Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris","volume":"25","author":"Baumgartner","year":"2003","journal-title":"Biotech Lett"},{"key":"2021033103281719200_CR2","volume-title":"High-yield protein production from Pichia pastoris yeast: a protocol for benchtop fermentation. Application Note","author":"Cino","year":"1999"},{"key":"2021033103281719200_CR3","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1002\/jmr.687","article-title":"Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production","volume":"18","author":"Daly","year":"2005","journal-title":"J Mol Recog"},{"key":"2021033103281719200_CR4","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1002\/ps.1119","article-title":"Insecticidal spider venom toxin fused to snowdrop lectin is toxic to the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) and the rice brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)","volume":"62","author":"Down","year":"2006","journal-title":"Pest Manag Sci"},{"key":"2021033103281719200_CR5","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1002\/ps.1833","article-title":"Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders","volume":"66","author":"Fitches","year":"2010","journal-title":"Pest Manag Sci"},{"key":"2021033103281719200_CR6","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1016\/j.jinsphys.2003.09.010","article-title":"Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion","volume":"50","author":"Fitches","year":"2004","journal-title":"J Insect Phys"},{"key":"2021033103281719200_CR7","doi-asserted-by":"publisher","first-page":"1213","DOI":"10.1016\/S0022-1910(98)00090-0","article-title":"A comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (Lacanobia oleracea)","volume":"44","author":"Fitches","year":"1998","journal-title":"J Insect Phys"},{"key":"2021033103281719200_CR8","doi-asserted-by":"publisher","first-page":"e39389","DOI":"10.1371\/journal.pone.0039389","article-title":"Fusion to snowdrop lectin dramatically enhances the oral activity of the insecticidal peptide \u03c9-hexatoxin-Hv1a by mediating its delivery to the central nervous system","volume":"7","author":"Fitches","year":"2012","journal-title":"PLoS One"},{"key":"2021033103281719200_CR9","doi-asserted-by":"publisher","first-page":"559","DOI":"10.1038\/nsb0797-559","article-title":"The structure of a novel insecticidal neurotoxin, \u03c9-atracotoxin-Hv1, from the venom of an Australian funnel web spider","volume":"4","author":"Fletcher","year":"1997","journal-title":"Nat Struct Biol"},{"key":"2021033103281719200_CR10","doi-asserted-by":"publisher","first-page":"191","DOI":"10.2217\/fmb.12.133","article-title":"Pichia pastoris: protein production host and model organism for biomedical research","volume":"8","author":"Gasser","year":"2013","journal-title":"Future Microbiol"},{"key":"2021033103281719200_CR11","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1385\/0-89603-421-6:81","article-title":"Generation of protease-deficient strains and their use in heterologous protein expression","volume":"103","author":"Gleeson","year":"1998","journal-title":"Methods Mol Biol"},{"key":"2021033103281719200_CR12","doi-asserted-by":"crossref","DOI":"10.1385\/0896034216","volume-title":"Pichia protocols","author":"Higgins","year":"1998"},{"key":"2021033103281719200_CR13","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1007\/s12010-007-0003-x","article-title":"Expression of recombinant proteins in Pichia Pastoris","volume":"142","author":"Li","year":"2007","journal-title":"Appl Biochem Biotech"},{"key":"2021033103281719200_CR14","doi-asserted-by":"crossref","first-page":"325","DOI":"10.2144\/000113672","article-title":"Extraction of genomic DNA from yeasts for PCR based applications","volume":"50","author":"L\u00f5oke","year":"2011","journal-title":"Biotechniques"},{"key":"2021033103281719200_CR15","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1007\/s10529-005-1007-7","article-title":"Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris","volume":"27","author":"Mansur","year":"2005","journal-title":"Biotech Letts"},{"key":"2021033103281719200_CR16","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1046\/j.1432-1327.1999.00749.x","article-title":"Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris\u2014Correct N terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide","volume":"265","author":"Raemaekers","year":"1999","journal-title":"Eur J Biochem"},{"key":"2021033103281719200_CR17","volume-title":"Molecular cloning: a laboratory manual","author":"Sambrook","year":"2001","edition":"3"},{"key":"2021033103281719200_CR18","doi-asserted-by":"publisher","first-page":"102","DOI":"10.1002\/bit.20318","article-title":"Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau","volume":"89","author":"Sinha","year":"2005","journal-title":"Biotechnol Bioeng"},{"key":"2021033103281719200_CR19","article-title":"Pichia optimization of protein expression","volume-title":"Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology","author":"Sreekrishna","year":"2012"},{"key":"2021033103281719200_CR20","doi-asserted-by":"publisher","first-page":"184","DOI":"10.1007\/s00253-005-0042-6","article-title":"Functional GNA expressed in E. coli with high efficiency and its effect on Ceratovacuna lanigera Zehntner","volume":"69","author":"Sulan","year":"2005","journal-title":"Appl Microbiol Biotech"},{"key":"2021033103281719200_CR21","doi-asserted-by":"publisher","first-page":"44133","DOI":"10.1074\/jbc.M404006200","article-title":"Scanning mutagenesis of \u03c9-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against invertebrate calcium channels","volume":"279","author":"Tedford","year":"2004","journal-title":"J Biol Chem"},{"key":"2021033103281719200_CR22","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1186\/1472-6750-6-18","article-title":"A fusion protein containing a lepidopteran-specific toxin from the South Indian red scorpion (Mesobuthus tamulus) and snowdrop lectin shows oral toxicity to target insects","volume":"6","author":"Trung","year":"2006","journal-title":"BMC Biotech"},{"key":"2021033103281719200_CR23","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1006\/prep.2000.1335","article-title":"Effect of copy number on the expression levels of Hepatitif B surface antigen in the methylotrophic yeast Pichia pastoris","volume":"21","author":"Vassileva","year":"2001","journal-title":"Protein Express Purif"},{"key":"2021033103281719200_CR24","doi-asserted-by":"publisher","first-page":"954","DOI":"10.1111\/j.1365-2672.2009.04279.x","article-title":"Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris","volume":"107","author":"Zhu","year":"2009","journal-title":"J Appl Micro"}],"container-title":["Journal of Industrial Microbiology and Biotechnology"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/link.springer.com\/content\/pdf\/10.1007\/s10295-014-1466-8.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/link.springer.com\/article\/10.1007\/s10295-014-1466-8\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/academic.oup.com\/jimb\/article-pdf\/41\/8\/1237\/36811224\/jimb1237.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/jimb\/article-pdf\/41\/8\/1237\/36811224\/jimb1237.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,7,13]],"date-time":"2023-07-13T23:39:30Z","timestamp":1689291570000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jimb\/article\/41\/8\/1237\/5995011"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,8,1]]},"references-count":24,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2014,8,1]]}},"URL":"https:\/\/doi.org\/10.1007\/s10295-014-1466-8","relation":{},"ISSN":["1476-5535","1367-5435"],"issn-type":[{"value":"1476-5535","type":"electronic"},{"value":"1367-5435","type":"print"}],"subject":[],"published-other":{"date-parts":[[2014,8,1]]},"published":{"date-parts":[[2014,8,1]]}}}