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Article

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure

1
CICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilhã, Portugal
2
Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal
3
UCIBIO–Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal
4
Laboratório de Fármaco-Toxicologia-UBIMedical, University of Beira Interior, 6201-284 Covilhã, Portugal
*
Author to whom correspondence should be addressed.
Academic Editor: Francesca Sanguedolce
Int. J. Mol. Sci. 2021, 22(18), 10012; https://doi.org/10.3390/ijms221810012
Received: 17 August 2021 / Revised: 8 September 2021 / Accepted: 10 September 2021 / Published: 16 September 2021
(This article belongs to the Collection Feature Papers in Molecular Biophysics)
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 (Tm) 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 Tm 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. View Full-Text
Keywords: circular dichroism; co-immunoprecipitation; prostate cancer; protein purification; STEAP1; thermal stability circular dichroism; co-immunoprecipitation; prostate cancer; protein purification; STEAP1; thermal stability
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MDPI and ACS Style

Barroca-Ferreira, J.; Cruz-Vicente, P.; Santos, M.F.A.; Rocha, S.M.; Santos-Silva, T.; Maia, C.J.; Passarinha, L.A. Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure. Int. J. Mol. Sci. 2021, 22, 10012. https://doi.org/10.3390/ijms221810012

AMA Style

Barroca-Ferreira J, Cruz-Vicente P, Santos MFA, Rocha SM, Santos-Silva T, Maia CJ, Passarinha LA. Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure. International Journal of Molecular Sciences. 2021; 22(18):10012. https://doi.org/10.3390/ijms221810012

Chicago/Turabian Style

Barroca-Ferreira, Jorge, Pedro Cruz-Vicente, Marino F.A. Santos, Sandra M. Rocha, Teresa Santos-Silva, Cláudio J. Maia, and Luís A. Passarinha 2021. "Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure" International Journal of Molecular Sciences 22, no. 18: 10012. https://doi.org/10.3390/ijms221810012

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