Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD + synthetase
The enzyme NAD + synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberc...
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| Veröffentlicht in: | Protein expression and purification 2002-08, Vol.25 (3), p.547-557 |
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| creator | Bellinzoni, Marco De Rossi, Edda Branzoni, Manuela Milano, Anna Peverali, Fiorenzo A Rizzi, Menico Riccardi, Giovanna |
| description | The enzyme NAD
+ synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of
Mycobacterium tuberculosis NAD
+ synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from
Escherichia coli strains to cultured High Five (
Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in
E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD
+ synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real
M. tuberculosis NAD
+ synthetase. These results enable us to obtain a purified product for structure–function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity. |
| doi_str_mv | 10.1016/S1046-5928(02)00041-4 |
| format | Article |
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+ synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of
Mycobacterium tuberculosis NAD
+ synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from
Escherichia coli strains to cultured High Five (
Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in
E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD
+ synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real
M. tuberculosis NAD
+ synthetase. These results enable us to obtain a purified product for structure–function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/S1046-5928(02)00041-4</identifier><identifier>PMID: 12182838</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Amide Synthases ; Amide Synthases - chemistry ; Amide Synthases - genetics ; Amide Synthases - isolation & purification ; Amide Synthases - metabolism ; Amino Acid Sequence ; Animals ; Biochemistry, Molecular Biology ; Bioinformatics ; Biological Physics ; Cellular Biology ; Chemical Sciences ; Computer Science ; Cristallography ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Escherichia coli - genetics ; Insecta ; Insecta - cytology ; Insecta - genetics ; Kinetics ; Life Sciences ; Molecular Sequence Data ; Mycobacterium tuberculosis ; Mycobacterium tuberculosis - enzymology ; Mycobacterium tuberculosis - genetics ; NAD ; NAD - metabolism ; Physics ; Recombinant Fusion Proteins ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Recombinant Fusion Proteins - metabolism ; Sequence Alignment ; Sequence Homology ; Structural Biology</subject><ispartof>Protein expression and purification, 2002-08, Vol.25 (3), p.547-557</ispartof><rights>2002 Elsevier Science (USA)</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c465t-dc7ec7073a840eb0bbaa1136d7b48f682f94948daa218fe2f193127ad8ccac4d3</citedby><cites>FETCH-LOGICAL-c465t-dc7ec7073a840eb0bbaa1136d7b48f682f94948daa218fe2f193127ad8ccac4d3</cites><orcidid>0000-0002-8887-6917</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1046592802000414$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,776,780,881,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12182838$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://pasteur.hal.science/pasteur-03257827$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Bellinzoni, Marco</creatorcontrib><creatorcontrib>De Rossi, Edda</creatorcontrib><creatorcontrib>Branzoni, Manuela</creatorcontrib><creatorcontrib>Milano, Anna</creatorcontrib><creatorcontrib>Peverali, Fiorenzo A</creatorcontrib><creatorcontrib>Rizzi, Menico</creatorcontrib><creatorcontrib>Riccardi, Giovanna</creatorcontrib><title>Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD + synthetase</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>The enzyme NAD
+ synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of
Mycobacterium tuberculosis NAD
+ synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from
Escherichia coli strains to cultured High Five (
Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in
E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD
+ synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real
M. tuberculosis NAD
+ synthetase. These results enable us to obtain a purified product for structure–function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.</description><subject>Amide Synthases</subject><subject>Amide Synthases - chemistry</subject><subject>Amide Synthases - genetics</subject><subject>Amide Synthases - isolation & purification</subject><subject>Amide Synthases - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biochemistry, Molecular Biology</subject><subject>Bioinformatics</subject><subject>Biological Physics</subject><subject>Cellular Biology</subject><subject>Chemical Sciences</subject><subject>Computer Science</subject><subject>Cristallography</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Insecta</subject><subject>Insecta - cytology</subject><subject>Insecta - genetics</subject><subject>Kinetics</subject><subject>Life Sciences</subject><subject>Molecular Sequence Data</subject><subject>Mycobacterium tuberculosis</subject><subject>Mycobacterium tuberculosis - enzymology</subject><subject>Mycobacterium tuberculosis - genetics</subject><subject>NAD</subject><subject>NAD - metabolism</subject><subject>Physics</subject><subject>Recombinant Fusion Proteins</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Sequence Alignment</subject><subject>Sequence Homology</subject><subject>Structural Biology</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcuO1DAQRS0EYoaBTwB5hUAQKDtO7KxQa3g0UgMLYG05ToUxSuKM7bQIX4_7IViyqodOVanuJeQxg1cMWP36KwNRF1XD1TPgzwFAsELcIZcMmroALpu7h_yMXJAHMf4EYKyG6j65YJwprkp1SYYtJgx-8D_8Ein-mgPG6Pz0ks5LcL2zJh0rM3UUp9_rmGtLjU1u79JKfU8_rda3uYHBLSNNS4vBLoOPLtLPm7f0BY3rlG4wmYgPyb3eDBEfneMV-f7-3bfrbbH78uHj9WZXWFFXqeisRCtBlkYJwBba1hjGyrqTrVB9rXjfiEaozpj8Ro-8Z03JuDSdstZY0ZVXpDjtvTGDnoMbTVi1N05vNzs9m5hwCRpKXknF5Z5l_umJn4O_XTAmPbpocRjMhFkWLTlkVSVksDqBNvgYA_Z_tzPQB1v00RZ90FwD10dbtMhzT84HlnbE7t_U2YcMvDkBmGXZOww6WoeTxc4FtEl33v3nxB8RMJ6h</recordid><startdate>20020801</startdate><enddate>20020801</enddate><creator>Bellinzoni, Marco</creator><creator>De Rossi, Edda</creator><creator>Branzoni, Manuela</creator><creator>Milano, Anna</creator><creator>Peverali, Fiorenzo A</creator><creator>Rizzi, Menico</creator><creator>Riccardi, Giovanna</creator><general>Elsevier Inc</general><general>Elsevier</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>1XC</scope><orcidid>https://orcid.org/0000-0002-8887-6917</orcidid></search><sort><creationdate>20020801</creationdate><title>Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD + synthetase</title><author>Bellinzoni, Marco ; De Rossi, Edda ; Branzoni, Manuela ; Milano, Anna ; Peverali, Fiorenzo A ; Rizzi, Menico ; Riccardi, Giovanna</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c465t-dc7ec7073a840eb0bbaa1136d7b48f682f94948daa218fe2f193127ad8ccac4d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Amide Synthases</topic><topic>Amide Synthases - chemistry</topic><topic>Amide Synthases - genetics</topic><topic>Amide Synthases - isolation & purification</topic><topic>Amide Synthases - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Biochemistry, Molecular Biology</topic><topic>Bioinformatics</topic><topic>Biological Physics</topic><topic>Cellular Biology</topic><topic>Chemical Sciences</topic><topic>Computer Science</topic><topic>Cristallography</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Insecta</topic><topic>Insecta - cytology</topic><topic>Insecta - genetics</topic><topic>Kinetics</topic><topic>Life Sciences</topic><topic>Molecular Sequence Data</topic><topic>Mycobacterium tuberculosis</topic><topic>Mycobacterium tuberculosis - enzymology</topic><topic>Mycobacterium tuberculosis - genetics</topic><topic>NAD</topic><topic>NAD - metabolism</topic><topic>Physics</topic><topic>Recombinant Fusion Proteins</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Sequence Alignment</topic><topic>Sequence Homology</topic><topic>Structural Biology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bellinzoni, Marco</creatorcontrib><creatorcontrib>De Rossi, Edda</creatorcontrib><creatorcontrib>Branzoni, Manuela</creatorcontrib><creatorcontrib>Milano, Anna</creatorcontrib><creatorcontrib>Peverali, Fiorenzo A</creatorcontrib><creatorcontrib>Rizzi, Menico</creatorcontrib><creatorcontrib>Riccardi, Giovanna</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>Hyper Article en Ligne (HAL)</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bellinzoni, Marco</au><au>De Rossi, Edda</au><au>Branzoni, Manuela</au><au>Milano, Anna</au><au>Peverali, Fiorenzo A</au><au>Rizzi, Menico</au><au>Riccardi, Giovanna</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD + synthetase</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2002-08-01</date><risdate>2002</risdate><volume>25</volume><issue>3</issue><spage>547</spage><epage>557</epage><pages>547-557</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>The enzyme NAD
+ synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of
Mycobacterium tuberculosis NAD
+ synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from
Escherichia coli strains to cultured High Five (
Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in
E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD
+ synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real
M. tuberculosis NAD
+ synthetase. These results enable us to obtain a purified product for structure–function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>12182838</pmid><doi>10.1016/S1046-5928(02)00041-4</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0002-8887-6917</orcidid></addata></record> |
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| subjects | Amide Synthases Amide Synthases - chemistry Amide Synthases - genetics Amide Synthases - isolation & purification Amide Synthases - metabolism Amino Acid Sequence Animals Biochemistry, Molecular Biology Bioinformatics Biological Physics Cellular Biology Chemical Sciences Computer Science Cristallography Electrophoresis, Polyacrylamide Gel Escherichia coli Escherichia coli - genetics Insecta Insecta - cytology Insecta - genetics Kinetics Life Sciences Molecular Sequence Data Mycobacterium tuberculosis Mycobacterium tuberculosis - enzymology Mycobacterium tuberculosis - genetics NAD NAD - metabolism Physics Recombinant Fusion Proteins Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Sequence Alignment Sequence Homology Structural Biology |
| title | Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD + synthetase |
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