Cargando…
The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli
We have recently shown that SmbP, the small metal-binding protein of Nitrosomonas europaea, can be employed as a fusion protein to express and purify recombinant proteins and peptides in Escherichia coli. SmbP increases solubility, allows simple, one-step purification through affinity chromatography...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929023/ https://www.ncbi.nlm.nih.gov/pubmed/35723324 http://dx.doi.org/10.3390/cimb44020038 |
_version_ | 1784670767228649472 |
---|---|
author | Martinez-Mora, Evelyn Arredondo-Espinoza, Eder Casillas-Vega, Nestor G. Cantu-Cardenas, Maria Elena Balderas-Renteria, Isaias Zarate, Xristo |
author_facet | Martinez-Mora, Evelyn Arredondo-Espinoza, Eder Casillas-Vega, Nestor G. Cantu-Cardenas, Maria Elena Balderas-Renteria, Isaias Zarate, Xristo |
author_sort | Martinez-Mora, Evelyn |
collection | PubMed |
description | We have recently shown that SmbP, the small metal-binding protein of Nitrosomonas europaea, can be employed as a fusion protein to express and purify recombinant proteins and peptides in Escherichia coli. 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 Buthus martensii Karsch. This peptide was expressed in Escherichia coli 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. |
format | Online Article Text |
id | pubmed-8929023 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-89290232022-06-04 The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli Martinez-Mora, Evelyn Arredondo-Espinoza, Eder Casillas-Vega, Nestor G. Cantu-Cardenas, Maria Elena Balderas-Renteria, Isaias Zarate, Xristo Curr Issues Mol Biol Article We have recently shown that SmbP, the small metal-binding protein of Nitrosomonas europaea, can be employed as a fusion protein to express and purify recombinant proteins and peptides in Escherichia coli. 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 Buthus martensii Karsch. This peptide was expressed in Escherichia coli 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. MDPI 2022-01-22 /pmc/articles/PMC8929023/ /pubmed/35723324 http://dx.doi.org/10.3390/cimb44020038 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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/). |
spellingShingle | Article Martinez-Mora, Evelyn Arredondo-Espinoza, Eder Casillas-Vega, Nestor G. Cantu-Cardenas, Maria Elena Balderas-Renteria, Isaias Zarate, Xristo The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title_full | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title_fullStr | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title_full_unstemmed | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title_short | The Small Metal-Binding Protein SmbP Improves the Expression and Purification of the Recombinant Antitumor-Analgesic Peptide from the Chinese Scorpion Buthus martensii Karsch in Escherichia coli |
title_sort | small metal-binding protein smbp improves the expression and purification of the recombinant antitumor-analgesic peptide from the chinese scorpion buthus martensii karsch in escherichia coli |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8929023/ https://www.ncbi.nlm.nih.gov/pubmed/35723324 http://dx.doi.org/10.3390/cimb44020038 |
work_keys_str_mv | AT martinezmoraevelyn thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT arredondoespinozaeder thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT casillasveganestorg thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT cantucardenasmariaelena thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT balderasrenteriaisaias thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT zaratexristo thesmallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT martinezmoraevelyn smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT arredondoespinozaeder smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT casillasveganestorg smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT cantucardenasmariaelena smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT balderasrenteriaisaias smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli AT zaratexristo smallmetalbindingproteinsmbpimprovestheexpressionandpurificationoftherecombinantantitumoranalgesicpeptidefromthechinesescorpionbuthusmartensiikarschinescherichiacoli |