Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen
► We describe the expression and purification of Na-GST-1 in the yeast Pichia pastoris. ► Recovery rates obtained were suitable for material for Phase 1 trial. ► A human hookworm vaccine is under development. The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu...
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| Veröffentlicht in: | Protein expression and purification 2012-06, Vol.83 (2), p.145-151 |
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| creator | Goud, Gaddam Narsa Deumic, Vehid Gupta, Richi Brelsford, Jill Zhan, Bin Gillespie, Portia Plieskatt, Jordan L. Tsao, Eric I. Hotez, Peter J. Bottazzi, Maria Elena |
| description | ► We describe the expression and purification of Na-GST-1 in the yeast Pichia pastoris. ► Recovery rates obtained were suitable for material for Phase 1 trial. ► A human hookworm vaccine is under development.
The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing. |
| doi_str_mv | 10.1016/j.pep.2012.03.013 |
| format | Article |
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The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2012.03.013</identifier><identifier>PMID: 22503665</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Albendazole ; Animals ; Antigens, Helminth - genetics ; Antigens, Helminth - isolation & purification ; Antigens, Helminth - metabolism ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Glutathione Transferase - genetics ; Glutathione Transferase - isolation & purification ; Glutathione Transferase - metabolism ; Helminth Proteins - genetics ; Helminth Proteins - isolation & purification ; Helminth Proteins - metabolism ; Hookworm ; Mebendazole ; Na-GST-1 ; Necator americanus ; Necator americanus - enzymology ; Pichia - genetics ; Pichia pastoris ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sabin Vaccine Institute ; Vaccine ; Vaccines</subject><ispartof>Protein expression and purification, 2012-06, Vol.83 (2), p.145-151</ispartof><rights>2012 Elsevier Inc.</rights><rights>Copyright © 2012 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-56cc04fe4a1014a43eb08f2cad6af5b087b97d7b358c8268898ad1328ef4b5a43</citedby><cites>FETCH-LOGICAL-c386t-56cc04fe4a1014a43eb08f2cad6af5b087b97d7b358c8268898ad1328ef4b5a43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.pep.2012.03.013$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22503665$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Goud, Gaddam Narsa</creatorcontrib><creatorcontrib>Deumic, Vehid</creatorcontrib><creatorcontrib>Gupta, Richi</creatorcontrib><creatorcontrib>Brelsford, Jill</creatorcontrib><creatorcontrib>Zhan, Bin</creatorcontrib><creatorcontrib>Gillespie, Portia</creatorcontrib><creatorcontrib>Plieskatt, Jordan L.</creatorcontrib><creatorcontrib>Tsao, Eric I.</creatorcontrib><creatorcontrib>Hotez, Peter J.</creatorcontrib><creatorcontrib>Bottazzi, Maria Elena</creatorcontrib><title>Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>► We describe the expression and purification of Na-GST-1 in the yeast Pichia pastoris. ► Recovery rates obtained were suitable for material for Phase 1 trial. ► A human hookworm vaccine is under development.
The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.</description><subject>Albendazole</subject><subject>Animals</subject><subject>Antigens, Helminth - genetics</subject><subject>Antigens, Helminth - isolation & purification</subject><subject>Antigens, Helminth - metabolism</subject><subject>Chromatography, Gel</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Glutathione Transferase - genetics</subject><subject>Glutathione Transferase - isolation & purification</subject><subject>Glutathione Transferase - metabolism</subject><subject>Helminth Proteins - genetics</subject><subject>Helminth Proteins - isolation & purification</subject><subject>Helminth Proteins - metabolism</subject><subject>Hookworm</subject><subject>Mebendazole</subject><subject>Na-GST-1</subject><subject>Necator americanus</subject><subject>Necator americanus - enzymology</subject><subject>Pichia - genetics</subject><subject>Pichia pastoris</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sabin Vaccine Institute</subject><subject>Vaccine</subject><subject>Vaccines</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAQhiMEoqXwAFyQj0UiwXYSxwunqioFqSqHlrPl2OOulyQOtrPQZ-SlmLCFI5w8M_r-f6z5i-IloxWjTLzdVTPMFaeMV7SuKKsfFceMbkRJebd5vNaNKNsNl0fFs5R2lDImaPu0OOK8pbUQ7XHx8-LHHCElH6Y3ZF6id97o_LvTkyVjGMAsg47Y6eE--USCI3kL5BqQCzgfIaJkWhK5G5as8xbFQG7KHPWUHESdgDByeq3Ly5vbkr1-R87IHINdzLpmLQ3uJxb2MIQZLHGrKxlAW4K-1ludgUQwYez9pKdMtiF8_R7iSPbaGI_LcOjvYHpePHF6SPDi4T0pvny4uD3_WF59vvx0fnZVmlqKXLbCGNo4aDQesdFNDT2VjhtthXYt1l2_6WzX1600kgspN1JbVnMJrulb5E-K04Mv_v3bAimr0ScDw6AnCEtSrGvrlkrM4P8oZZ3gtJM1ouyAmhhSiuDUHP2o4z1CKyfUTmHcao1b0Vph3Kh59WC_9CPYv4o_-SLw_gAA3mPvIapkPEwGrMeLZmWD_4f9L9HSvk0</recordid><startdate>201206</startdate><enddate>201206</enddate><creator>Goud, Gaddam Narsa</creator><creator>Deumic, Vehid</creator><creator>Gupta, Richi</creator><creator>Brelsford, Jill</creator><creator>Zhan, Bin</creator><creator>Gillespie, Portia</creator><creator>Plieskatt, Jordan L.</creator><creator>Tsao, Eric I.</creator><creator>Hotez, Peter J.</creator><creator>Bottazzi, Maria Elena</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>201206</creationdate><title>Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen</title><author>Goud, Gaddam Narsa ; Deumic, Vehid ; Gupta, Richi ; Brelsford, Jill ; Zhan, Bin ; Gillespie, Portia ; Plieskatt, Jordan L. ; Tsao, Eric I. ; Hotez, Peter J. ; Bottazzi, Maria Elena</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-56cc04fe4a1014a43eb08f2cad6af5b087b97d7b358c8268898ad1328ef4b5a43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Albendazole</topic><topic>Animals</topic><topic>Antigens, Helminth - genetics</topic><topic>Antigens, Helminth - isolation & purification</topic><topic>Antigens, Helminth - metabolism</topic><topic>Chromatography, Gel</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Glutathione Transferase - genetics</topic><topic>Glutathione Transferase - isolation & purification</topic><topic>Glutathione Transferase - metabolism</topic><topic>Helminth Proteins - genetics</topic><topic>Helminth Proteins - isolation & purification</topic><topic>Helminth Proteins - metabolism</topic><topic>Hookworm</topic><topic>Mebendazole</topic><topic>Na-GST-1</topic><topic>Necator americanus</topic><topic>Necator americanus - enzymology</topic><topic>Pichia - genetics</topic><topic>Pichia pastoris</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sabin Vaccine Institute</topic><topic>Vaccine</topic><topic>Vaccines</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Goud, Gaddam Narsa</creatorcontrib><creatorcontrib>Deumic, Vehid</creatorcontrib><creatorcontrib>Gupta, Richi</creatorcontrib><creatorcontrib>Brelsford, Jill</creatorcontrib><creatorcontrib>Zhan, Bin</creatorcontrib><creatorcontrib>Gillespie, Portia</creatorcontrib><creatorcontrib>Plieskatt, Jordan L.</creatorcontrib><creatorcontrib>Tsao, Eric I.</creatorcontrib><creatorcontrib>Hotez, Peter J.</creatorcontrib><creatorcontrib>Bottazzi, Maria Elena</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Goud, Gaddam Narsa</au><au>Deumic, Vehid</au><au>Gupta, Richi</au><au>Brelsford, Jill</au><au>Zhan, Bin</au><au>Gillespie, Portia</au><au>Plieskatt, Jordan L.</au><au>Tsao, Eric I.</au><au>Hotez, Peter J.</au><au>Bottazzi, Maria Elena</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2012-06</date><risdate>2012</risdate><volume>83</volume><issue>2</issue><spage>145</spage><epage>151</epage><pages>145-151</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>► We describe the expression and purification of Na-GST-1 in the yeast Pichia pastoris. ► Recovery rates obtained were suitable for material for Phase 1 trial. ► A human hookworm vaccine is under development.
The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>22503665</pmid><doi>10.1016/j.pep.2012.03.013</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1046-5928 |
| ispartof | Protein expression and purification, 2012-06, Vol.83 (2), p.145-151 |
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| language | eng |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Albendazole Animals Antigens, Helminth - genetics Antigens, Helminth - isolation & purification Antigens, Helminth - metabolism Chromatography, Gel Chromatography, High Pressure Liquid Electrophoresis, Polyacrylamide Gel Glutathione Transferase - genetics Glutathione Transferase - isolation & purification Glutathione Transferase - metabolism Helminth Proteins - genetics Helminth Proteins - isolation & purification Helminth Proteins - metabolism Hookworm Mebendazole Na-GST-1 Necator americanus Necator americanus - enzymology Pichia - genetics Pichia pastoris Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sabin Vaccine Institute Vaccine Vaccines |
| title | Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): A production process developed for a lead candidate recombinant hookworm vaccine antigen |
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