Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli
Bromelain, a member of cysteine proteases, is found abundantly in pineapple ( Ananas comosus ), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims t...
Gespeichert in:
| Veröffentlicht in: | The Protein Journal 2021-06, Vol.40 (3), p.406-418 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 418 |
|---|---|
| container_issue | 3 |
| container_start_page | 406 |
| container_title | The Protein Journal |
| container_volume | 40 |
| creator | Razali, Rafida Budiman, Cahyo Kamaruzaman, Khairul Azfar Subbiah, Vijay Kumar |
| description | Bromelain, a member of cysteine proteases, is found abundantly in pineapple (
Ananas comosus
), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using
Escherichia coli
host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into
Escherichia coli
BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni
2+
-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine
p
-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg
−1
and inhibited by a cysteine protease inhibitor, E-64 (IC
50
of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (
k
ca
t
/K
M
) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10
–2
µM
−1
s
−1
while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain. |
| doi_str_mv | 10.1007/s10930-021-09974-9 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_proqu</sourceid><recordid>TN_cdi_proquest_miscellaneous_2501265948</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A716527538</galeid><sourcerecordid>A716527538</sourcerecordid><originalsourceid>FETCH-LOGICAL-c442t-8133b006b065e9e174be45b363051f88feffb505731d9d17c8292b4f35590383</originalsourceid><addsrcrecordid>eNp9kUtv1TAQhSMEog_4AyyQJTbdpPgZx8tyuQWkVq2ge8txxq2rxA52ItHy5_G9t1CBKuSFR-PvHM_oVNUbgo8JxvJ9JlgxXGNKaqyU5LV6Vu0TIVnNFWfPtzWtWdvKveog51uMaaskfVntMSYJo1zsVz-_xWHpBkDrH1OCnH0MyIQercxshrvZW3SZ4gRp9pBRdGgV-xjqi2n2o7-HHn0FG8fOBxNm9CHFEQbjA3KlQucfKbr0Acw0Ff_SXWd7A8nbG2-QjYN_Vb1wZsjw-uE-rK5O11erz_XZxacvq5Oz2nJO57oljHUYNx1uBCggknfARccahgVxbevAuU7gsjfpVU-kbamiHXdMCIVZyw6ro53tlOL3BfKsR58tDIMJEJesqcCENkLxDfruH_Q2LimU4QrFqWCCMfxIXZsBtA8uzsnYjak-kaQRVIrtt8dPUOX0MHobAzhf-n8J6E5gU8w5gdNT8qNJd5pgvQlc7wLXJXC9DVyrInr7MPHSjdD_kfxOuABsB-TyFK4hPa70H9tfhvizzw</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>2542535330</pqid></control><display><type>article</type><title>Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli</title><source>MEDLINE</source><source>SpringerLink Journals - AutoHoldings</source><creator>Razali, Rafida ; Budiman, Cahyo ; Kamaruzaman, Khairul Azfar ; Subbiah, Vijay Kumar</creator><creatorcontrib>Razali, Rafida ; Budiman, Cahyo ; Kamaruzaman, Khairul Azfar ; Subbiah, Vijay Kumar</creatorcontrib><description>Bromelain, a member of cysteine proteases, is found abundantly in pineapple (
Ananas comosus
), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using
Escherichia coli
host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into
Escherichia coli
BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni
2+
-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine
p
-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg
−1
and inhibited by a cysteine protease inhibitor, E-64 (IC
50
of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (
k
ca
t
/K
M
) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10
–2
µM
−1
s
−1
while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.</description><identifier>ISSN: 1572-3887</identifier><identifier>EISSN: 1573-4943</identifier><identifier>EISSN: 1875-8355</identifier><identifier>DOI: 10.1007/s10930-021-09974-9</identifier><identifier>PMID: 33713245</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Acetic acid ; Ananas - enzymology ; Ananas - genetics ; Animal Anatomy ; Biochemistry ; Bioorganic Chemistry ; Bromelains - biosynthesis ; Bromelains - chemistry ; Bromelains - genetics ; Bromelains - isolation & purification ; Catalysis ; Catalytic activity ; Chemistry ; Chemistry and Materials Science ; Circular dichroism ; Codon ; Commercialization ; Cysteine ; Cysteine proteinase ; Dichroism ; E coli ; Escherichia coli ; Ethylenediaminetetraacetic acid ; Ethylenediaminetetraacetic acids ; Fruit bromelain ; Gene Expression ; Genetic aspects ; Histology ; Metal ions ; Morphology ; Optimization ; Organic Chemistry ; pH effects ; Pineapples ; Plant Proteins - biosynthesis ; Plant Proteins - chemistry ; Plant Proteins - genetics ; Plant Proteins - isolation & purification ; Protease inhibitors ; Proteinase inhibitors ; Recombinant Proteins - biosynthesis ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Spectroscopy ; Thioredoxin</subject><ispartof>The Protein Journal, 2021-06, Vol.40 (3), p.406-418</ispartof><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021</rights><rights>COPYRIGHT 2021 Springer</rights><rights>The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c442t-8133b006b065e9e174be45b363051f88feffb505731d9d17c8292b4f35590383</citedby><cites>FETCH-LOGICAL-c442t-8133b006b065e9e174be45b363051f88feffb505731d9d17c8292b4f35590383</cites><orcidid>0000-0002-0384-1580 ; 0000-0002-2052-9572</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10930-021-09974-9$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10930-021-09974-9$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33713245$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Razali, Rafida</creatorcontrib><creatorcontrib>Budiman, Cahyo</creatorcontrib><creatorcontrib>Kamaruzaman, Khairul Azfar</creatorcontrib><creatorcontrib>Subbiah, Vijay Kumar</creatorcontrib><title>Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli</title><title>The Protein Journal</title><addtitle>Protein J</addtitle><addtitle>Protein J</addtitle><description>Bromelain, a member of cysteine proteases, is found abundantly in pineapple (
Ananas comosus
), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using
Escherichia coli
host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into
Escherichia coli
BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni
2+
-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine
p
-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg
−1
and inhibited by a cysteine protease inhibitor, E-64 (IC
50
of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (
k
ca
t
/K
M
) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10
–2
µM
−1
s
−1
while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.</description><subject>Acetic acid</subject><subject>Ananas - enzymology</subject><subject>Ananas - genetics</subject><subject>Animal Anatomy</subject><subject>Biochemistry</subject><subject>Bioorganic Chemistry</subject><subject>Bromelains - biosynthesis</subject><subject>Bromelains - chemistry</subject><subject>Bromelains - genetics</subject><subject>Bromelains - isolation & purification</subject><subject>Catalysis</subject><subject>Catalytic activity</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Circular dichroism</subject><subject>Codon</subject><subject>Commercialization</subject><subject>Cysteine</subject><subject>Cysteine proteinase</subject><subject>Dichroism</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Ethylenediaminetetraacetic acid</subject><subject>Ethylenediaminetetraacetic acids</subject><subject>Fruit bromelain</subject><subject>Gene Expression</subject><subject>Genetic aspects</subject><subject>Histology</subject><subject>Metal ions</subject><subject>Morphology</subject><subject>Optimization</subject><subject>Organic Chemistry</subject><subject>pH effects</subject><subject>Pineapples</subject><subject>Plant Proteins - biosynthesis</subject><subject>Plant Proteins - chemistry</subject><subject>Plant Proteins - genetics</subject><subject>Plant Proteins - isolation & purification</subject><subject>Protease inhibitors</subject><subject>Proteinase inhibitors</subject><subject>Recombinant Proteins - biosynthesis</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Spectroscopy</subject><subject>Thioredoxin</subject><issn>1572-3887</issn><issn>1573-4943</issn><issn>1875-8355</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp9kUtv1TAQhSMEog_4AyyQJTbdpPgZx8tyuQWkVq2ge8txxq2rxA52ItHy5_G9t1CBKuSFR-PvHM_oVNUbgo8JxvJ9JlgxXGNKaqyU5LV6Vu0TIVnNFWfPtzWtWdvKveog51uMaaskfVntMSYJo1zsVz-_xWHpBkDrH1OCnH0MyIQercxshrvZW3SZ4gRp9pBRdGgV-xjqi2n2o7-HHn0FG8fOBxNm9CHFEQbjA3KlQucfKbr0Acw0Ff_SXWd7A8nbG2-QjYN_Vb1wZsjw-uE-rK5O11erz_XZxacvq5Oz2nJO57oljHUYNx1uBCggknfARccahgVxbevAuU7gsjfpVU-kbamiHXdMCIVZyw6ro53tlOL3BfKsR58tDIMJEJesqcCENkLxDfruH_Q2LimU4QrFqWCCMfxIXZsBtA8uzsnYjak-kaQRVIrtt8dPUOX0MHobAzhf-n8J6E5gU8w5gdNT8qNJd5pgvQlc7wLXJXC9DVyrInr7MPHSjdD_kfxOuABsB-TyFK4hPa70H9tfhvizzw</recordid><startdate>20210601</startdate><enddate>20210601</enddate><creator>Razali, Rafida</creator><creator>Budiman, Cahyo</creator><creator>Kamaruzaman, Khairul Azfar</creator><creator>Subbiah, Vijay Kumar</creator><general>Springer US</general><general>Springer</general><general>Springer Nature B.V</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>3V.</scope><scope>7QL</scope><scope>7TK</scope><scope>7TM</scope><scope>7U7</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PDBOC</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-0384-1580</orcidid><orcidid>https://orcid.org/0000-0002-2052-9572</orcidid></search><sort><creationdate>20210601</creationdate><title>Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli</title><author>Razali, Rafida ; Budiman, Cahyo ; Kamaruzaman, Khairul Azfar ; Subbiah, Vijay Kumar</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c442t-8133b006b065e9e174be45b363051f88feffb505731d9d17c8292b4f35590383</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Acetic acid</topic><topic>Ananas - enzymology</topic><topic>Ananas - genetics</topic><topic>Animal Anatomy</topic><topic>Biochemistry</topic><topic>Bioorganic Chemistry</topic><topic>Bromelains - biosynthesis</topic><topic>Bromelains - chemistry</topic><topic>Bromelains - genetics</topic><topic>Bromelains - isolation & purification</topic><topic>Catalysis</topic><topic>Catalytic activity</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Circular dichroism</topic><topic>Codon</topic><topic>Commercialization</topic><topic>Cysteine</topic><topic>Cysteine proteinase</topic><topic>Dichroism</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Ethylenediaminetetraacetic acid</topic><topic>Ethylenediaminetetraacetic acids</topic><topic>Fruit bromelain</topic><topic>Gene Expression</topic><topic>Genetic aspects</topic><topic>Histology</topic><topic>Metal ions</topic><topic>Morphology</topic><topic>Optimization</topic><topic>Organic Chemistry</topic><topic>pH effects</topic><topic>Pineapples</topic><topic>Plant Proteins - biosynthesis</topic><topic>Plant Proteins - chemistry</topic><topic>Plant Proteins - genetics</topic><topic>Plant Proteins - isolation & purification</topic><topic>Protease inhibitors</topic><topic>Proteinase inhibitors</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Spectroscopy</topic><topic>Thioredoxin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Razali, Rafida</creatorcontrib><creatorcontrib>Budiman, Cahyo</creatorcontrib><creatorcontrib>Kamaruzaman, Khairul Azfar</creatorcontrib><creatorcontrib>Subbiah, Vijay Kumar</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Materials Science Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Protein Journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Razali, Rafida</au><au>Budiman, Cahyo</au><au>Kamaruzaman, Khairul Azfar</au><au>Subbiah, Vijay Kumar</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli</atitle><jtitle>The Protein Journal</jtitle><stitle>Protein J</stitle><addtitle>Protein J</addtitle><date>2021-06-01</date><risdate>2021</risdate><volume>40</volume><issue>3</issue><spage>406</spage><epage>418</epage><pages>406-418</pages><issn>1572-3887</issn><eissn>1573-4943</eissn><eissn>1875-8355</eissn><abstract>Bromelain, a member of cysteine proteases, is found abundantly in pineapple (
Ananas comosus
), and it has a myriad of versatile applications. However, attempts to produce recombinant bromelain for commercialization purposes are challenging due to its expressibility and solubility. This study aims to express recombinant fruit bromelain from MD2 pineapple (MD2Bro; accession no: OAY85858.1) in soluble and active forms using
Escherichia coli
host cell. The gene encoding MD2Bro was codon-optimized, synthesized, and subsequently ligated into pET-32b( +) for further transformation into
Escherichia coli
BL21-CodonPlus(DE3). Under this strategy, the expressed MD2Bro was in a fusion form with thioredoxin (Trx) tag at its N-terminal (Trx-MD2Bro). The result showed that Trx-MD2Bro was successfully expressed in fully soluble form. The protein was successfully purified using single-step Ni
2+
-NTA chromatography and confirmed to be in proper folds based on the circular dichroism spectroscopy analysis. The purified Trx-MD2Bro was confirmed to be catalytically active against N-carbobenzoxyglycine
p
-nitrophenyl ester (N-CBZ-Gly-pNP) with a specific activity of 6.13 ± 0.01 U mg
−1
and inhibited by a cysteine protease inhibitor, E-64 (IC
50
of 74.38 ± 1.65 nM). Furthermore, the catalytic efficiency (
k
ca
t
/K
M
) Trx-MD2Bro was calculated to be at 5.64 ± 0.02 × 10
–2
µM
−1
s
−1
while the optimum temperature and pH were at 50 °C and pH 6.0, respectively. Furthermore, the catalytic activity of Trx-MD2Bro was also affected by ethylenediaminetetraacetic acid (EDTA) or metal ions. Altogether it is proposed that the combination of codon optimization and the use of an appropriate vector are important in the production of a soluble and actively stable recombinant bromelain.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>33713245</pmid><doi>10.1007/s10930-021-09974-9</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-0384-1580</orcidid><orcidid>https://orcid.org/0000-0002-2052-9572</orcidid></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1572-3887 |
| ispartof | The Protein Journal, 2021-06, Vol.40 (3), p.406-418 |
| issn | 1572-3887 1573-4943 1875-8355 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_2501265948 |
| source | MEDLINE; SpringerLink Journals - AutoHoldings |
| subjects | Acetic acid Ananas - enzymology Ananas - genetics Animal Anatomy Biochemistry Bioorganic Chemistry Bromelains - biosynthesis Bromelains - chemistry Bromelains - genetics Bromelains - isolation & purification Catalysis Catalytic activity Chemistry Chemistry and Materials Science Circular dichroism Codon Commercialization Cysteine Cysteine proteinase Dichroism E coli Escherichia coli Ethylenediaminetetraacetic acid Ethylenediaminetetraacetic acids Fruit bromelain Gene Expression Genetic aspects Histology Metal ions Morphology Optimization Organic Chemistry pH effects Pineapples Plant Proteins - biosynthesis Plant Proteins - chemistry Plant Proteins - genetics Plant Proteins - isolation & purification Protease inhibitors Proteinase inhibitors Recombinant Proteins - biosynthesis Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Spectroscopy Thioredoxin |
| title | Soluble Expression and Catalytic Properties of Codon-Optimized Recombinant Bromelain from MD2 Pineapple in Escherichia coli |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-09T12%3A48%3A36IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_proqu&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Soluble%20Expression%20and%20Catalytic%20Properties%20of%20Codon-Optimized%20Recombinant%20Bromelain%20from%20MD2%20Pineapple%20in%20Escherichia%20coli&rft.jtitle=The%20Protein%20Journal&rft.au=Razali,%20Rafida&rft.date=2021-06-01&rft.volume=40&rft.issue=3&rft.spage=406&rft.epage=418&rft.pages=406-418&rft.issn=1572-3887&rft.eissn=1573-4943&rft_id=info:doi/10.1007/s10930-021-09974-9&rft_dat=%3Cgale_proqu%3EA716527538%3C/gale_proqu%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2542535330&rft_id=info:pmid/33713245&rft_galeid=A716527538&rfr_iscdi=true |