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 2020–Operational Programme Competitiveness and Internationalisation in Axis I–Strengthening 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 Luís A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from FCT–Fundação para a Ciência 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 Bioeconomy–i4HB (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-β-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

Trends in protein-based biosensor assemblies for drug screening and pharmaceutical kinetic studies

The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters.info:eu-repo/semantics/publishedVersio

Evaluation of the growth factors VEGF-a and VEGF-B in the vitreous and serum of patients with macular and retinal vascular diseases

VEGF-A and VEGF-B are proangiogenic and key regulating factors for blood vessel growth. This study aims to compare VEGF-A and VEGF-B levels in the serum and vitreous of patients with neovascular pathology versus non-neovascular pathology. Our findings showed vitreous VEGF-A and VEGF-B levels increased in patients with neovascular disease, with higher levels of VEGF-A compared to VEGF-B (p ≤ .05). In the diabetic retinopathy (DR) group, higher vitreous VEGF-A or VEGF-B were found in proliferative diabetic retinopathy (PDR) than in non-PDR. The strong correlation between VEGF-A and VEGF-B demonstrates a simultaneous pathological increase of cytokines (p < .001), suggesting besides VEGF-A, VEGF-B is another contributor to ocular pathologies involving angiogenesis. There was no correlation between vitreous and serum VEGF-A or VEGF-B; however, a correlation between vitreous (VEGF-A or VEGF-B) and macular volume (p < .05) in DR patients was found. Targeting VEGF-A and VEGF-B in macular and retinal vascular diseases, involving neovascularization, may improve treatment outcomes.POCI-01-0145-FEDER-007491info:eu-repo/semantics/publishedVersio

Vascular endothelial growth factors and placenta growth factor in retinal vasculopathies: Current research and future perspectives

Vision loss due to disease or degeneration of the eye (retina, choroid, retinal veins, or macula) is a leading cause of blindness worldwide. In most cases, vision-threatening ocular diseases are accompanied by abnormal changes in the vasculature of the eye, especially the retina, and these conditions are collectively referred to as retinal vasculopathies. Impaired blood supply or hypoxia stimulates angiogenesis in the vascular and non-vascular sections of the eye, which results in neovascularization, leading to conditions such as diabetic retinopathy or age-related macular degeneration. Studies show that vascular endothelial growth factors: VEGF-A, VEGF-B, and placental growth factor (PlGF) are elevated in these diseases, and hence, these factors could be used as markers for disease prognosis and therapy. In this review, we discuss the function of these growth factors in normal development and disease, with focus on ocular disorders and emphasize the importance of accurately determining their levels in the vitreous and serum of patients for correct diagnosis and therapy.info:eu-repo/semantics/publishedVersio

A review of preparation, characterization and emerging applications

The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020–Operational Programme Competitiveness and Internationalisation in Axis I–Strengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491 ), from FCT –Fundação para a Ciência e Tecnologia. Publisher Copyright: © 2023 The AuthorsGellan gum, a microbial exopolysaccharide, is biodegradable and has potential to fill several key roles in many fields from food to pharmacy, biomedicine and tissue engineering. In order to improve the physicochemical and biological properties of gellan gum, some researchers take advantage of numerous hydroxyl groups and the free carboxyl present in each repeating unit. As a result, design and development of gellan-based materials have advanced significantly. The goal of this review is to provide a summary of the most recent, high-quality research trends that have used gellan gum as a polymeric component in the design of numerous cutting-edge materials with applications in various fields.publishersversionpublishe

Pichia pastoris: a recombinant microfactory for antibodies and human membrane proteins

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.info:eu-repo/semantics/publishedVersio

Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.info:eu-repo/semantics/publishedVersio