The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i>
We have recently shown that SmbP, the small metal-binding protein of <i>Nitrosomonas europaea</i>, can be employed as a fusion protein to express and purify recombinant proteins and peptides in <i>Escherichia coli</i>. SmbP increases solubility, allows simple, one-step purifi...
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MDPI AG
2022-01-01
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| Series: | Current Issues in Molecular Biology |
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| Online Access: | https://www.mdpi.com/1467-3045/44/2/38 |
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| author | Evelyn Martinez-Mora Eder Arredondo-Espinoza Nestor G. Casillas-Vega Maria Elena Cantu-Cardenas Isaias Balderas-Renteria Xristo Zarate |
| author_facet | Evelyn Martinez-Mora Eder Arredondo-Espinoza Nestor G. Casillas-Vega Maria Elena Cantu-Cardenas Isaias Balderas-Renteria Xristo Zarate |
| author_sort | Evelyn Martinez-Mora |
| collection | DOAJ |
| description | We have recently shown that SmbP, the small metal-binding protein of <i>Nitrosomonas europaea</i>, can be employed as a fusion protein to express and purify recombinant proteins and peptides in <i>Escherichia coli</i>. SmbP increases solubility, allows simple, one-step purification through affinity chromatography, and provides superior final yields due to its low molecular weight. In this work, we report for the first time the use of SmbP to produce a recombinant peptide with anticancer activity: the antitumor-analgesic peptide (BmK-AGAP), a neurotoxin isolated from the venom of the Chinese scorpion <i>Buthus martensii</i> Karsch. This peptide was expressed in <i>Escherichia coli</i> SHuffle for correct, cytoplasmic, disulfide bond formation and tagged with SmbP at the N-terminus to improve its solubility and allow purification using immobilized metal affinity chromatography. SmbP_BmK-AGAP was found in the soluble fraction of the cell lysate. After purification and removal of SmbP by digestion with enterokinase, 1.8 mg of pure and highly active rBmK-AGAP was obtained per liter of cell culture. rBmK-AGAP exhibited antiproliferative activity on the MCF-7 cancer cell line, with a half-maximal inhibitory concentration value of 7.24 μM. Based on these results, we considered SmbP to be a suitable carrier protein for the production of recombinant, biologically active BmK-AGAP. We propose that SmbP should be an attractive fusion protein for the expression and purification of additional recombinant proteins or peptides that display anticancer activities. |
| first_indexed | 2024-03-09T22:17:30Z |
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| language | English |
| last_indexed | 2024-03-09T22:17:30Z |
| publishDate | 2022-01-01 |
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| series | Current Issues in Molecular Biology |
| spelling | doaj.art-ac18bfc6748a485da2e0213d0000ae102023-11-23T19:20:52ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452022-01-0144255055810.3390/cimb44020038The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i>Evelyn Martinez-Mora0Eder Arredondo-Espinoza1Nestor G. Casillas-Vega2Maria Elena Cantu-Cardenas3Isaias Balderas-Renteria4Xristo Zarate5Facultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, Av. Universidad s/n, Cd. Universitaria, San Nicolas de los Garza 66455, MexicoFacultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, Av. Universidad s/n, Cd. Universitaria, San Nicolas de los Garza 66455, MexicoDepartamento de Patologia Clinica, Hospital Universitario Dr. Jose Eleuterio Gonzalez, Universidad Autonoma de Nuevo Leon, Monterrey 64460, MexicoFacultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, Av. Universidad s/n, Cd. Universitaria, San Nicolas de los Garza 66455, MexicoFacultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, Av. Universidad s/n, Cd. Universitaria, San Nicolas de los Garza 66455, MexicoFacultad de Ciencias Quimicas, Universidad Autonoma de Nuevo Leon, Av. Universidad s/n, Cd. Universitaria, San Nicolas de los Garza 66455, MexicoWe have recently shown that SmbP, the small metal-binding protein of <i>Nitrosomonas europaea</i>, can be employed as a fusion protein to express and purify recombinant proteins and peptides in <i>Escherichia coli</i>. SmbP increases solubility, allows simple, one-step purification through affinity chromatography, and provides superior final yields due to its low molecular weight. In this work, we report for the first time the use of SmbP to produce a recombinant peptide with anticancer activity: the antitumor-analgesic peptide (BmK-AGAP), a neurotoxin isolated from the venom of the Chinese scorpion <i>Buthus martensii</i> Karsch. This peptide was expressed in <i>Escherichia coli</i> SHuffle for correct, cytoplasmic, disulfide bond formation and tagged with SmbP at the N-terminus to improve its solubility and allow purification using immobilized metal affinity chromatography. SmbP_BmK-AGAP was found in the soluble fraction of the cell lysate. After purification and removal of SmbP by digestion with enterokinase, 1.8 mg of pure and highly active rBmK-AGAP was obtained per liter of cell culture. rBmK-AGAP exhibited antiproliferative activity on the MCF-7 cancer cell line, with a half-maximal inhibitory concentration value of 7.24 μM. Based on these results, we considered SmbP to be a suitable carrier protein for the production of recombinant, biologically active BmK-AGAP. We propose that SmbP should be an attractive fusion protein for the expression and purification of additional recombinant proteins or peptides that display anticancer activities.https://www.mdpi.com/1467-3045/44/2/38SmbPsmall metal-binding proteinBmK-AGAP<i>Escherichia coli</i>recombinant peptidesanticancer activity |
| spellingShingle | Evelyn Martinez-Mora Eder Arredondo-Espinoza Nestor G. Casillas-Vega Maria Elena Cantu-Cardenas Isaias Balderas-Renteria Xristo Zarate The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> Current Issues in Molecular Biology SmbP small metal-binding protein BmK-AGAP <i>Escherichia coli</i> recombinant peptides anticancer activity |
| title | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> |
| title_full | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> |
| title_fullStr | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> |
| title_full_unstemmed | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> |
| title_short | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion <i>Buthus martensii</i> Karsch in <i>Escherichia coli</i> |
| title_sort | small metal binding protein smbp improves the expression and purification of the recombinant antitumor analgesic peptide from the chinese scorpion i buthus martensii i karsch in i escherichia coli i |
| topic | SmbP small metal-binding protein BmK-AGAP <i>Escherichia coli</i> recombinant peptides anticancer activity |
| url | https://www.mdpi.com/1467-3045/44/2/38 |
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