52 research outputs found
A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates
Funding Information: The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020âOperational Programme Competitiveness and Internationalisation in Axis IâStrengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491), Jorge Barroca-Ferreira's and Ana M. Gonçalves's individual PhD Fellowships (SFRH/BD/130068/2017 and SFRH/BD/147519/2019, respectively), and LuĂs A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from FCTâFundação para a CiĂȘncia e Tecnologia. This work was also supported by the Health Sciences Research Centre CICS-UBI (UIDB/00709/2020 and UIDP/00709/2020), the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomyâi4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Publisher Copyright: © 2022The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-ÎČ-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.publishersversionpublishe
Growth responses and accumulation of soluble sugars in Inga marginata Wild. (Fabaceae) subjected to flooding under contrasting light conditions
Vegetative development and content of calcium, potassium, and sodium in watermelon under salinity stress on organic substrates
Anålise da erosividade das chuvas associada aos padrÔes de precipitação pluvial na região de Ribeirão das Lajes (RJ)
PrevalĂȘncia de enteroparisitos na populaçao urbana de SĂŁo TomĂ© - RN - estudo comparativo de um decĂȘnio
Reactivation of latent bovine herpesvirus type 5 in cattle with polioencephalomalacia induced by ammonium sulphate
FRUIT MORPHOLOGY AND PRODUCTIVITY OF BABASSU PALMS IN NORTHERN MINAS GERAIS STATE, BRAZIL1
Manganese accumulation and its relation to "eucalyptus shoot blight in the Vale do Rio Doce"
- âŠ