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Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein

The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellula...

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Autores principales: Kusuma, Sri Agung Fitri, Fadhlillah, Muhammad, Rostinawati, Tina, Maisyarah, Intan Timur, Syafitri, Raden Indah Puspita, Subroto, Toto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10378983/
https://www.ncbi.nlm.nih.gov/pubmed/37504457
http://dx.doi.org/10.3390/gels9070578
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author Kusuma, Sri Agung Fitri
Fadhlillah, Muhammad
Rostinawati, Tina
Maisyarah, Intan Timur
Syafitri, Raden Indah Puspita
Subroto, Toto
author_facet Kusuma, Sri Agung Fitri
Fadhlillah, Muhammad
Rostinawati, Tina
Maisyarah, Intan Timur
Syafitri, Raden Indah Puspita
Subroto, Toto
author_sort Kusuma, Sri Agung Fitri
collection PubMed
description The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method.
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spelling pubmed-103789832023-07-29 Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein Kusuma, Sri Agung Fitri Fadhlillah, Muhammad Rostinawati, Tina Maisyarah, Intan Timur Syafitri, Raden Indah Puspita Subroto, Toto Gels Article The production and purification of recombinant proteins are crucial to acquiring pure MPT64 protein. Due to the fact that protein epitopes may undergo conformational changes during purification, this study, therefore, investigated an effective rapid purification method to produce highly intracellular pure MPT64 protein without causing conformational changes in the epitope under denaturing conditions. MPT64 was isolated from E. coli and electrophoresed using gel SDS-PAGE. Then, the desired protein bands were excised and purified with two methods: electroelution and passive elution. The isolated protein was identified via peptide mass fingerprinting using MALDI-TOF MS and reacted with IgG anti-MPT64, and the cross-reactivity of the isolated protein with IgY anti-MPT64 was confirmed using Western blot. The results show that both of these methods produced pure MPT64 protein, and the MPT64 protein was confirmed based on the MALDI-TOF MS results. Neither of these two methods resulted in epitope changes in the MPT64 protein so it could react specifically with both antibodies. The yield of MPT64 protein was higher with electroelution (2030 ± 41 µg/mL) than with passive elution (179.5 ± 7.5 µg/mL). Thus, it can be inferred that the electroelution method is a more effective method of purifying MPT64 protein and maintaining its epitope than the passive elution method. MDPI 2023-07-14 /pmc/articles/PMC10378983/ /pubmed/37504457 http://dx.doi.org/10.3390/gels9070578 Text en © 2023 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
Kusuma, Sri Agung Fitri
Fadhlillah, Muhammad
Rostinawati, Tina
Maisyarah, Intan Timur
Syafitri, Raden Indah Puspita
Subroto, Toto
Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title_full Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title_fullStr Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title_full_unstemmed Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title_short Gel Protein Extraction’s Impact on Conformational Epitopes of Linear Non-Tagged MPT64 Protein
title_sort gel protein extraction’s impact on conformational epitopes of linear non-tagged mpt64 protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10378983/
https://www.ncbi.nlm.nih.gov/pubmed/37504457
http://dx.doi.org/10.3390/gels9070578
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