Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis
Cry11 proteins are toxic to , the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shufflin...
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| Veröffentlicht in: | International journal of molecular sciences 2023-05, Vol.24 (10), p.9079 |
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| creator | Suárez-Barrera, Miguel O Herrera-Pineda, Diego F Rondón-Villarreal, Paola Pinzón-Reyes, Efraín Hernando Ochoa, Rodrigo Visser, Lydia Rueda-Forero, Nohora Juliana |
| description | Cry11 proteins are toxic to
, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of
and subjected to median-lethal concentration (LC
) tests on first-instar larvae of
. LC
analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL
), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against
This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines. |
| doi_str_mv | 10.3390/ijms24109079 |
| format | Article |
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, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of
and subjected to median-lethal concentration (LC
) tests on first-instar larvae of
. LC
analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL
), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against
This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms24109079</identifier><identifier>PMID: 37240424</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Aedes - genetics ; Aedes - metabolism ; Amino acids ; Analysis ; Animals ; Bacillus thuringiensis - genetics ; Bacillus thuringiensis - metabolism ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacterial Proteins - toxicity ; Cancer ; Cell viability ; Colorectal carcinoma ; Cytotoxicity ; Disease control ; DNA shuffling ; Endotoxins - genetics ; Endotoxins - metabolism ; Endotoxins - toxicity ; Hemolysin Proteins - genetics ; Hemolysin Proteins - metabolism ; Insects ; Larva - genetics ; Larva - metabolism ; Molecular dynamics ; Mosquito Vectors ; Mutagenesis ; Mutants ; Mutation ; Phenylalanine ; Protein structure ; Proteins ; Site-directed mutagenesis ; Tertiary structure ; Toxicity ; Toxins ; Tryptophan ; Tumor cell lines ; Vector-borne diseases ; Vectors (Biology) ; Zika Virus - metabolism ; Zika Virus Infection</subject><ispartof>International journal of molecular sciences, 2023-05, Vol.24 (10), p.9079</ispartof><rights>COPYRIGHT 2023 MDPI AG</rights><rights>2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2023 by the authors. 2023</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c437t-d4f2a46156a4f7e4730ed888f3692d6903b0767ea2fc97732342fa4cb9b633303</cites><orcidid>0000-0003-1189-0603 ; 0000-0001-9258-8101 ; 0000-0003-0734-2196 ; 0000-0002-0276-6646 ; 0000-0003-4503-3482 ; 0000-0001-8209-3885</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10219489/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10219489/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,883,27907,27908,53774,53776</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37240424$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Suárez-Barrera, Miguel O</creatorcontrib><creatorcontrib>Herrera-Pineda, Diego F</creatorcontrib><creatorcontrib>Rondón-Villarreal, Paola</creatorcontrib><creatorcontrib>Pinzón-Reyes, Efraín Hernando</creatorcontrib><creatorcontrib>Ochoa, Rodrigo</creatorcontrib><creatorcontrib>Visser, Lydia</creatorcontrib><creatorcontrib>Rueda-Forero, Nohora Juliana</creatorcontrib><title>Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis</title><title>International journal of molecular sciences</title><addtitle>Int J Mol Sci</addtitle><description>Cry11 proteins are toxic to
, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of
and subjected to median-lethal concentration (LC
) tests on first-instar larvae of
. LC
analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL
), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against
This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.</description><subject>Aedes - genetics</subject><subject>Aedes - metabolism</subject><subject>Amino acids</subject><subject>Analysis</subject><subject>Animals</subject><subject>Bacillus thuringiensis - genetics</subject><subject>Bacillus thuringiensis - metabolism</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacterial Proteins - toxicity</subject><subject>Cancer</subject><subject>Cell viability</subject><subject>Colorectal carcinoma</subject><subject>Cytotoxicity</subject><subject>Disease control</subject><subject>DNA shuffling</subject><subject>Endotoxins - genetics</subject><subject>Endotoxins - metabolism</subject><subject>Endotoxins - toxicity</subject><subject>Hemolysin Proteins - genetics</subject><subject>Hemolysin Proteins - metabolism</subject><subject>Insects</subject><subject>Larva - genetics</subject><subject>Larva - metabolism</subject><subject>Molecular dynamics</subject><subject>Mosquito Vectors</subject><subject>Mutagenesis</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Phenylalanine</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Site-directed mutagenesis</subject><subject>Tertiary structure</subject><subject>Toxicity</subject><subject>Toxins</subject><subject>Tryptophan</subject><subject>Tumor cell lines</subject><subject>Vector-borne diseases</subject><subject>Vectors (Biology)</subject><subject>Zika Virus - metabolism</subject><subject>Zika Virus Infection</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNptkt1rFDEUxQdRbK2--SwBX3zotjcfM5k8ybJWW6i0SPscMplkzTqTbJOMuP-92W5btiKBJNz7OyfccKrqPYYTSgWcutWYCMMggIsX1SFmhMwAGv5y735QvUlpBUAoqcXr6oBywoARdlj9ugl_nEZfTDZxdF5lFzwKFi3iBmP0fcoqmx5dx5CN8wlddVk5Xyq3yfkl-nHPq6Hok1t6pHyPLnJCizCut9Jdc162TXLpbfXKqiGZdw_nUXX79exmcT67vPp2sZhfzjSjPM96ZoliDa4bxSw3jFMwfdu2ljaC9I0A2gFvuFHEasE5JZQRq5juRNdQSoEeVZ93vuupG02vjc9RDXId3ajiRgbl5POOdz_lMvyWGAgWrBXF4dODQwx3k0lZji5pMwzKmzAlSVoCgJm4Rz_-g67CFMvEW6q41Qxgj1qqwUjnbSgP662pnPOaMFIXv0Kd_Icqqzej08Eb60r9meB4J9AxpBSNfRoSg9ymQ-6no-Af9j_mCX6MA_0LOe20Dg</recordid><startdate>20230522</startdate><enddate>20230522</enddate><creator>Suárez-Barrera, Miguel O</creator><creator>Herrera-Pineda, Diego F</creator><creator>Rondón-Villarreal, Paola</creator><creator>Pinzón-Reyes, Efraín Hernando</creator><creator>Ochoa, Rodrigo</creator><creator>Visser, Lydia</creator><creator>Rueda-Forero, Nohora Juliana</creator><general>MDPI AG</general><general>MDPI</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>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-1189-0603</orcidid><orcidid>https://orcid.org/0000-0001-9258-8101</orcidid><orcidid>https://orcid.org/0000-0003-0734-2196</orcidid><orcidid>https://orcid.org/0000-0002-0276-6646</orcidid><orcidid>https://orcid.org/0000-0003-4503-3482</orcidid><orcidid>https://orcid.org/0000-0001-8209-3885</orcidid></search><sort><creationdate>20230522</creationdate><title>Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis</title><author>Suárez-Barrera, Miguel O ; Herrera-Pineda, Diego F ; Rondón-Villarreal, Paola ; Pinzón-Reyes, Efraín Hernando ; Ochoa, Rodrigo ; Visser, Lydia ; Rueda-Forero, Nohora Juliana</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c437t-d4f2a46156a4f7e4730ed888f3692d6903b0767ea2fc97732342fa4cb9b633303</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Aedes - genetics</topic><topic>Aedes - metabolism</topic><topic>Amino acids</topic><topic>Analysis</topic><topic>Animals</topic><topic>Bacillus thuringiensis - genetics</topic><topic>Bacillus thuringiensis - metabolism</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Bacterial Proteins - toxicity</topic><topic>Cancer</topic><topic>Cell viability</topic><topic>Colorectal carcinoma</topic><topic>Cytotoxicity</topic><topic>Disease control</topic><topic>DNA shuffling</topic><topic>Endotoxins - genetics</topic><topic>Endotoxins - metabolism</topic><topic>Endotoxins - toxicity</topic><topic>Hemolysin Proteins - genetics</topic><topic>Hemolysin Proteins - metabolism</topic><topic>Insects</topic><topic>Larva - genetics</topic><topic>Larva - metabolism</topic><topic>Molecular dynamics</topic><topic>Mosquito Vectors</topic><topic>Mutagenesis</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Phenylalanine</topic><topic>Protein structure</topic><topic>Proteins</topic><topic>Site-directed mutagenesis</topic><topic>Tertiary structure</topic><topic>Toxicity</topic><topic>Toxins</topic><topic>Tryptophan</topic><topic>Tumor cell lines</topic><topic>Vector-borne diseases</topic><topic>Vectors (Biology)</topic><topic>Zika Virus - metabolism</topic><topic>Zika Virus Infection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Suárez-Barrera, Miguel O</creatorcontrib><creatorcontrib>Herrera-Pineda, Diego F</creatorcontrib><creatorcontrib>Rondón-Villarreal, Paola</creatorcontrib><creatorcontrib>Pinzón-Reyes, Efraín Hernando</creatorcontrib><creatorcontrib>Ochoa, Rodrigo</creatorcontrib><creatorcontrib>Visser, Lydia</creatorcontrib><creatorcontrib>Rueda-Forero, Nohora Juliana</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>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Research Library (Corporate)</collection><collection>Publicly Available Content Database</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 China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>International journal of molecular sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Suárez-Barrera, Miguel O</au><au>Herrera-Pineda, Diego F</au><au>Rondón-Villarreal, Paola</au><au>Pinzón-Reyes, Efraín Hernando</au><au>Ochoa, Rodrigo</au><au>Visser, Lydia</au><au>Rueda-Forero, Nohora Juliana</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis</atitle><jtitle>International journal of molecular sciences</jtitle><addtitle>Int J Mol Sci</addtitle><date>2023-05-22</date><risdate>2023</risdate><volume>24</volume><issue>10</issue><spage>9079</spage><pages>9079-</pages><issn>1422-0067</issn><issn>1661-6596</issn><eissn>1422-0067</eissn><abstract>Cry11 proteins are toxic to
, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of
and subjected to median-lethal concentration (LC
) tests on first-instar larvae of
. LC
analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL
), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against
This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>37240424</pmid><doi>10.3390/ijms24109079</doi><orcidid>https://orcid.org/0000-0003-1189-0603</orcidid><orcidid>https://orcid.org/0000-0001-9258-8101</orcidid><orcidid>https://orcid.org/0000-0003-0734-2196</orcidid><orcidid>https://orcid.org/0000-0002-0276-6646</orcidid><orcidid>https://orcid.org/0000-0003-4503-3482</orcidid><orcidid>https://orcid.org/0000-0001-8209-3885</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Aedes - genetics Aedes - metabolism Amino acids Analysis Animals Bacillus thuringiensis - genetics Bacillus thuringiensis - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacterial Proteins - toxicity Cancer Cell viability Colorectal carcinoma Cytotoxicity Disease control DNA shuffling Endotoxins - genetics Endotoxins - metabolism Endotoxins - toxicity Hemolysin Proteins - genetics Hemolysin Proteins - metabolism Insects Larva - genetics Larva - metabolism Molecular dynamics Mosquito Vectors Mutagenesis Mutants Mutation Phenylalanine Protein structure Proteins Site-directed mutagenesis Tertiary structure Toxicity Toxins Tryptophan Tumor cell lines Vector-borne diseases Vectors (Biology) Zika Virus - metabolism Zika Virus Infection |
| title | Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis |
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