{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:21:11Z","timestamp":1760235671724,"version":"build-2065373602"},"reference-count":62,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2021,9,16]],"date-time":"2021-09-16T00:00:00Z","timestamp":1631750400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 \u00b0C was determined, confirming STEAP1 stability in the purification buffer. A predominant \u03b1-helical structure was identified, ensuring the protein\u2019s 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.<\/jats:p>","DOI":"10.3390\/ijms221810012","type":"journal-article","created":{"date-parts":[[2021,9,16]],"date-time":"2021-09-16T21:38:12Z","timestamp":1631828292000},"page":"10012","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0142-5719","authenticated-orcid":false,"given":"Jorge","family":"Barroca-Ferreira","sequence":"first","affiliation":[{"name":"CICS-UBI\u2013Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO\u2013Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8552-6180","authenticated-orcid":false,"given":"Pedro","family":"Cruz-Vicente","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2013Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO\u2013Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8205-1041","authenticated-orcid":false,"given":"Marino F. A.","family":"Santos","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO\u2013Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4686-2410","authenticated-orcid":false,"given":"Sandra M.","family":"Rocha","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2013Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]},{"given":"Teresa","family":"Santos-Silva","sequence":"additional","affiliation":[{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO\u2013Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5658-5445","authenticated-orcid":false,"given":"Cl\u00e1udio J.","family":"Maia","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2013Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6910-7576","authenticated-orcid":false,"given":"Lu\u00eds A.","family":"Passarinha","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2013Health Sciences Research Centre, University of Beira Interior, 6201-506 Covilh\u00e3, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO\u2013Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"Laborat\u00f3rio de F\u00e1rmaco-Toxicologia-UBIMedical, University of Beira Interior, 6201-284 Covilh\u00e3, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"14523","DOI":"10.1073\/pnas.96.25.14523","article-title":"STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors","volume":"96","author":"Hubert","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"222","DOI":"10.2174\/1568009617666170427103732","article-title":"Targeting STEAP1 protein in human cancer: Current trends and future challenges","volume":"18","author":"Pais","year":"2018","journal-title":"Curr. Cancer Drug Targets"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2617","DOI":"10.1016\/j.yexcr.2013.07.025","article-title":"Six-transmembrane epithelial antigen of the prostate-1 plays a role for in vivo tumor growth via intercellular communication","volume":"319","author":"Yamamoto","year":"2013","journal-title":"Exp. Cell Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1158\/1541-7786.MCR-11-0524","article-title":"STEAP1 is associated with the invasive and oxidative stress phenotype of Ewing tumors","volume":"10","author":"Grunewald","year":"2012","journal-title":"Mol. Cancer Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9502","DOI":"10.1074\/jbc.RA120.013690","article-title":"Cryo-EM structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1","volume":"295","author":"Oosterheert","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"6673","DOI":"10.1021\/acs.biochem.6b00610","article-title":"Six-transmembrane epithelial antigen of prostate 1 (STEAP1) has a single b heme and is capable of reducing metal ion complexes and oxygen","volume":"55","author":"Kim","year":"2016","journal-title":"Biochemistry"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1111\/his.12226","article-title":"STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate carcinoma","volume":"63","author":"Drigo","year":"2013","journal-title":"Histopathology"},{"key":"ref_8","first-page":"23","article-title":"STEAP1 is overexpressed in prostate cancer and prostatic intraepithelial neoplasia lesions, and it is positively associated with Gleason score","volume":"32","author":"Gomes","year":"2014","journal-title":"Urol. Oncol. Semin. Orig. Investig."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1002\/pros.22601","article-title":"Six transmembrane epithelial antigen of the prostate 1 is down-regulated by sex hormones in prostate cancer","volume":"73","author":"Gomes","year":"2013","journal-title":"Prostate"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1007\/s12032-018-1100-0","article-title":"Knockdown of STEAP1 inhibits cell growth and induces apoptosis in LNCaP prostate cancer cells counteracting the effect of androgens","volume":"35","author":"Gomes","year":"2018","journal-title":"Med. Oncol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1517","DOI":"10.2967\/jnumed.118.222844","article-title":"Imaging metastatic castration-resistant prostate cancer patients with 89Zr-DFO-MSTP2109A anti-STEAP1 antibody","volume":"60","author":"Carrasquillo","year":"2019","journal-title":"J. Nucl. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3518","DOI":"10.1200\/JCO.19.00646","article-title":"Phase I Study of DSTP3086S, an Antibody-Drug Conjugate Targeting Six-Transmembrane Epithelial Antigen of Prostate 1, in Metastatic Castration-Resistant Prostate Cancer","volume":"37","author":"Danila","year":"2019","journal-title":"J. Clin. Oncol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Schober, S.J., Thiede, M., Gassmann, H., Prexler, C., Xue, B., Schirmer, D., Wohlleber, D., Stein, S., Gr\u00fcnewald, T.G., and Busch, D.H. (2020). MHC Class I-Restricted TCR-Transgenic CD4+ T Cells Against STEAP1 Mediate Local Tumor Control of Ewing Sarcoma In Vivo. Cells, 9.","DOI":"10.3390\/cells9071581"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1016\/j.biopha.2019.01.012","article-title":"Inhibition of mouse RM-1 prostate cancer and B16F10 melanoma by the fusion protein of HSP65 & STEAP1 186\u2013193","volume":"111","author":"Chen","year":"2019","journal-title":"Biomed. Pharmacother."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1111\/boc.201200027","article-title":"The STEAP protein family: Versatile oxidoreductases and targets for cancer immunotherapy with overlapping and distinct cellular functions","volume":"104","author":"Grunewald","year":"2012","journal-title":"Biol. Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.bbrc.2012.10.123","article-title":"STEAP1 is overexpressed in cancers: A promising therapeutic target","volume":"429","author":"Moreaux","year":"2012","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Rocha, S.M., Barroca-Ferreira, J., Passarinha, L.A., Socorro, S., and Maia, C.J. (2021). The Usefulness of STEAP Proteins in Prostate Cancer Clinical Practice. Prostate Cancer, Exon Publications.","DOI":"10.36255\/exonpublications.prostatecancer.steap.2021"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"7410","DOI":"10.1073\/pnas.0801318105","article-title":"Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle","volume":"105","author":"Sendamarai","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4337","DOI":"10.1038\/s41467-018-06817-7","article-title":"Cryo-EM structures of human STEAP4 reveal mechanism of iron (III) reduction","volume":"9","author":"Oosterheert","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3840","DOI":"10.1007\/s12010-015-1551-0","article-title":"Evaluation of MutS and Mut+ Pichia pastoris Strains for Membrane-Bound Catechol-O-Methyltransferase Biosynthesis","volume":"175","author":"Pedro","year":"2015","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1007\/s10337-015-2970-3","article-title":"Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography","volume":"78","author":"Pedro","year":"2015","journal-title":"Chromatographia"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4635","DOI":"10.1007\/s00253-021-11367-y","article-title":"Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter","volume":"105","author":"Duarte","year":"2021","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Smith, S.M. (2017). Strategies for the purification of membrane proteins. Protein Chromatography, Springer.","DOI":"10.1007\/978-1-4939-6412-3_21"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1139\/bcb-2015-0143","article-title":"Current strategies for protein production and purification enabling membrane protein structural biology","volume":"94","author":"Pandey","year":"2016","journal-title":"Biochem. Cell Biol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Tomaz, C.T. (2017). Hydrophobic interaction chromatography. Liquid Chromatography, Elsevier.","DOI":"10.1016\/B978-0-12-805393-5.00007-5"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"858","DOI":"10.1002\/bmc.1377","article-title":"Assessment of COMT isolation by HIC using a dual salt system and low temperature","volume":"24","author":"Nunes","year":"2010","journal-title":"Biomed. Chromatogr."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1002\/bmc.961","article-title":"Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt system","volume":"22","author":"Sousa","year":"2008","journal-title":"Biomed. Chromatogr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/BF01838161","article-title":"Zur lehre von der wirkung der salze","volume":"25","author":"Hofmeister","year":"1888","journal-title":"Arch. Exp. Pathol. Pharmakol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/S0021-9673(01)93737-9","article-title":"Salt-promoted adsorption chromatography","volume":"510","author":"Porath","year":"1990","journal-title":"J. Chromatogr. A"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.chroma.2007.06.002","article-title":"A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography","volume":"1177","author":"Passarinha","year":"2008","journal-title":"J. Chromatogr. A"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1002\/bmc.779","article-title":"Comparative study on the interaction of recombinant human soluble catechol-O-methyltransferase on some hydrophobic adsorbents","volume":"21","author":"Passarinha","year":"2007","journal-title":"Biomed. Chromatogr."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"O\u2019Connor, B.F., and Cummins, P.M. (2017). Hydrophobic Interaction Chromatography. Protein Chromatography, Springer.","DOI":"10.1007\/978-1-4939-6412-3_18"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/S1570-0232(03)00080-1","article-title":"Hydrophobic interaction chromatography of proteins: II. Binding capacity, recovery and mass transfer properties","volume":"790","author":"Hahn","year":"2003","journal-title":"J. Chromatogr. B"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/S0021-9673(02)01077-4","article-title":"Hydrophobic interaction chromatography of proteins: I. Comparison of selectivity","volume":"972","author":"Machold","year":"2002","journal-title":"J. Chromatogr. A"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Rothnie, A.J. (2016). Detergent-free membrane protein purification. Heterologous Expression of Membrane Proteins, Springer.","DOI":"10.1007\/978-1-4939-3637-3_16"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.procbio.2016.10.013","article-title":"Separation of human Fab fragments by negative chromatography on \u03c9-aminohexyl-and poly (ethyleneimine)-agarose","volume":"52","author":"Carmignotto","year":"2017","journal-title":"Process Biochem."},{"key":"ref_37","first-page":"163","article-title":"Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography","volume":"1017","author":"Carmignotto","year":"2016","journal-title":"J. Chromatogr. B"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.ab.2010.01.011","article-title":"A unified method for purification of basic proteins","volume":"400","author":"Adhikari","year":"2010","journal-title":"Anal. Biochem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.chroma.2004.10.047","article-title":"Polyethylene glycol increases purification and recovery, alters retention behavior in flow-through chromatography of hemoglobin","volume":"1059","author":"Lu","year":"2004","journal-title":"J. Chromatogr. A"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.chroma.2015.08.056","article-title":"Negative chromatography purification of hepatitis B virus-like particles using poly (oligo (ethylene glycol) methacrylate) grafted cationic adsorbent","volume":"1415","author":"Lee","year":"2015","journal-title":"J. Chromatogr. A"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1016\/j.jchromb.2013.09.030","article-title":"Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties","volume":"940","author":"Gurramkonda","year":"2013","journal-title":"J. Chromatogr. B"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1016\/j.procbio.2014.02.018","article-title":"Negative chromatography: Progress, applications and future perspectives","volume":"49","author":"Lee","year":"2014","journal-title":"Process Biochem."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/cpcb.3","article-title":"Immunoprecipitation","volume":"Volume 71","author":"Bonifacino","year":"2016","journal-title":"Current Protocol in Cell Biology"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Lee, C. (2007). Coimmunoprecipitation assay. Circadian Rhythms, Springer.","DOI":"10.1007\/978-1-59745-257-1_31"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1992","DOI":"10.1016\/j.mex.2019.09.001","article-title":"Immunoprecipitation methods to identify S-glutathionylation in target proteins","volume":"6","author":"Butturini","year":"2019","journal-title":"MethodsX"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1478","DOI":"10.1002\/pmic.201100563","article-title":"History of protein\u2013protein interactions: From egg-white to complex networks","volume":"12","author":"Braun","year":"2012","journal-title":"Proteomics"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-019-49145-6","article-title":"Identification of new binding proteins of focal adhesion kinase using immunoprecipitation and mass spectrometry","volume":"9","author":"Nguyen","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Lin, J.-S., Ma, L.-S., and Lai, E.-M. (2013). Systematic dissection of the agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0067647"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Lin, J.-S., and Lai, E.-M. (2017). Protein\u2013Protein Interactions: Co-Immunoprecipitation. Bacterial Protein Secretion Systems, Springer.","DOI":"10.1007\/978-1-4939-7033-9_17"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Kaboord, B., and Perr, M. (2008). Isolation of proteins and protein complexes by immunoprecipitation. 2D PAGE: Sample Preparation and Fractionation, Springer.","DOI":"10.1007\/978-1-60327-064-9_27"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1002\/0471140864.ps2809s79","article-title":"Analysis of protein stability and ligand interactions by thermal shift assay","volume":"79","author":"Huynh","year":"2015","journal-title":"Curr. Protoc. Protein Sci."},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Luan, C.-H., Light, S.H., Dunne, S.F., and Anderson, W.F. (2014). Ligand screening using fluorescence thermal shift analysis (FTS). Structural Genomics and Drug Discovery, Springer.","DOI":"10.1007\/978-1-4939-0354-2_20"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/j.ab.2006.07.027","article-title":"Thermofluor-based high-throughput stability optimization of proteins for structural studies","volume":"357","author":"Ericsson","year":"2006","journal-title":"Anal. Biochem."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1107\/S1744309112012912","article-title":"Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes","volume":"68","author":"Geders","year":"2012","journal-title":"Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.pep.2011.10.001","article-title":"Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin","volume":"81","author":"Santos","year":"2012","journal-title":"Protein Expr. Purif."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.pep.2013.08.002","article-title":"Optimization of protein purification and characterization using Thermofluor screens","volume":"91","author":"Boivin","year":"2013","journal-title":"Protein Expr. Purif."},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Ramos, J., Muthukumaran, J., Freire, F., Paquete-Ferreira, J., Otrelo-Cardoso, A.R., Svergun, D., Panjkovich, A., and Santos-Silva, T. (2019). Shedding light on the interaction of human anti-apoptotic Bcl-2 protein with ligands through biophysical and in silico studies. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20040860"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1002\/2211-5463.12617","article-title":"Human aldehyde oxidase (hAOX 1): Structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms","volume":"9","author":"Mota","year":"2019","journal-title":"FEBS Open Bio"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"2751","DOI":"10.1016\/j.chempr.2019.07.008","article-title":"Electronic circular dichroism spectroscopy of proteins","volume":"5","author":"Rogers","year":"2019","journal-title":"Chemistry"},{"key":"ref_60","doi-asserted-by":"crossref","unstructured":"Pignataro, M.F., Herrera, M.G., and Dodero, V.I. (2020). Evaluation of Peptide\/Protein Self-Assembly and Aggregation by Spectroscopic Methods. Molecules, 25.","DOI":"10.3390\/molecules25204854"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"4859","DOI":"10.1039\/C5CS00084J","article-title":"Circular dichroism spectroscopy of membrane proteins","volume":"45","author":"Miles","year":"2016","journal-title":"Chem. Soc. Rev."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/18\/10012\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:00:49Z","timestamp":1760166049000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/18\/10012"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,16]]},"references-count":62,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["ijms221810012"],"URL":"https:\/\/doi.org\/10.3390\/ijms221810012","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2021,9,16]]}}}