Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure
Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sephar...
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| Format: | Article |
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MDPI AG
2021-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/18/10012 |
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| author | Jorge Barroca-Ferreira Pedro Cruz-Vicente Marino F. A. Santos Sandra M. Rocha Teresa Santos-Silva Cláudio J. Maia Luís A. Passarinha |
| author_facet | Jorge Barroca-Ferreira Pedro Cruz-Vicente Marino F. A. Santos Sandra M. Rocha Teresa Santos-Silva Cláudio J. Maia Luís A. Passarinha |
| author_sort | Jorge Barroca-Ferreira |
| collection | DOAJ |
| description | Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (<i>T</i>m) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A <i>T</i>m of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment. |
| first_indexed | 2024-03-10T07:35:29Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T07:35:29Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-1f9f37ced5184c8ea0ec7a38e68b0deb2023-11-22T13:31:07ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-09-0122181001210.3390/ijms221810012Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation ProcedureJorge Barroca-Ferreira0Pedro Cruz-Vicente1Marino F. A. Santos2Sandra M. Rocha3Teresa Santos-Silva4Cláudio J. Maia5Luís A. Passarinha6CICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalCICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalAssociate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, PortugalCICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalAssociate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, PortugalCICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalCICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, PortugalBackground: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (<i>T</i>m) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A <i>T</i>m of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.https://www.mdpi.com/1422-0067/22/18/10012circular dichroismco-immunoprecipitationprostate cancerprotein purificationSTEAP1thermal stability |
| spellingShingle | Jorge Barroca-Ferreira Pedro Cruz-Vicente Marino F. A. Santos Sandra M. Rocha Teresa Santos-Silva Cláudio J. Maia Luís A. Passarinha Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure International Journal of Molecular Sciences circular dichroism co-immunoprecipitation prostate cancer protein purification STEAP1 thermal stability |
| title | Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure |
| title_full | Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure |
| title_fullStr | Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure |
| title_full_unstemmed | Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure |
| title_short | Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure |
| title_sort | enhanced stability of detergent free human native steap1 protein from neoplastic prostate cancer cells upon an innovative isolation procedure |
| topic | circular dichroism co-immunoprecipitation prostate cancer protein purification STEAP1 thermal stability |
| url | https://www.mdpi.com/1422-0067/22/18/10012 |
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