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The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Analytical Chemistry".

Deadline for manuscript submissions: closed (31 July 2020) | Viewed by 28906

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Special Issue Editor

Prof. Dr. Paolo Iadarola

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Department of Biology and Biotechnology “L. Spallanzani”, Universita degli Studi di Pavia, Pavia, Italy
Interests: purification and characterization of enzymes and structural proteins; investigation of the proteome of different tissues/fluids by using the conventional methods of proteomics/metabolomics
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Special Issue Information

Dear Colleagues,

A general protocol for protein purification involves the isolation of these macromolecules from a wide range of sources, which include animal organs, plants, bacteria, viruses, and other sources. To purify the protein of interest from a crude extract, appropriate techniques must be combined in a logical way, to minimize the number of steps and to maximize yields. Although the production of proteins through recombinant DNA techniques has made their purification simpler, not all challenges have been removed. Contaminants, as well as problems related to solubility, structural integrity, and biological activity, still exist. To achieve the purity level that fits the desired criteria, highly specific procedures are needed. The key to the successful purification of a protein lies in the successful application of the most recent technological advancements made in the field of biosciences. The purification step is essential and preliminary to an accurate characterization of a protein that, because of the intrinsic features of each protein, involve the use of multiple techniques. These include ELISA for the identification of the protein; UV and VIS spectrophotometry for the calculation of the protein concentration; SDS-PAGE, SEC, and MS for the determination of the molecular weight; chemical and/or enzymatic digestion in combination with HPLC and MS for peptide mapping; the Edman chemistry for the analysis of the primary sequence; and CD, X-ray diffraction, and NMR for the determination of secondary and tertiary structures.
The aim of this Special Issue is to attract contributions on all aspects of protein purification and characterization, with special emphasis on the application of the most sophisticated technologies that allow for addressing the purification processes characterized by peculiar difficulties.

Prof. Dr. Paolo Iadarola
Guest Editor

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Keywords

Proteins with and without catalytic activity
1- and 2-DE
ELISA
HPLC/UPLC
MS and MS/MS

Published Papers (7 papers)

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Research

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14 pages, 4260 KiB

Open AccessArticle

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers

by Giuliana Ferrante, Rossana Rossi, Giovanna Cilluffo, Dario Di Silvestre, Andrea Brambilla, Antonella De Palma, Chiara Villa, Velia Malizia, Rosalia Gagliardo, Yvan Torrente, Giovanni Corsello, Giovanni Viegi, Pierluigi Mauri and Stefania La Grutta

Molecules 2021, 26(5), 1258; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26051258 - 26 Feb 2021

Cited by 2 | Viewed by 2055

Abstract

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3–5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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14 pages, 1274 KiB

Open AccessArticle

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma

by Elisa Maffioli, Zhenze Jiang, Simona Nonnis, Armando Negri, Valentina Romeo, Christopher B. Lietz, Vivian Hook, Giuseppe Ristagno, Giuseppe Baselli, Erik B. Kistler, Federico Aletti, Anthony J. O’Donoghue and Gabriella Tedeschi

Molecules 2020, 25(18), 4071; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25184071 - 06 Sep 2020

Cited by 9 | Viewed by 2920

Abstract

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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18 pages, 2987 KiB

Open AccessArticle

Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

by Muzammeer Mansor, Jameel R. Al-Obaidi, Nurain Nadiah Jaafar, Intan Hakimah Ismail, Atiqah Farah Zakaria, Mohd Azri Zainal Abidin, Jinap Selamat, Son Radu and Nuzul Noorahya Jambari

Molecules 2020, 25(11), 2625; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25112625 - 05 Jun 2020

Cited by 10 | Viewed by 3761

Abstract

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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13 pages, 2050 KiB

Open AccessArticle

Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat α_s1-Casein

by Aliah Zannierah Mohsin, Rashidah Sukor, Jinap Selamat, Anis Shobirin Meor Hussin, Intan Hakimah Ismail, Nuzul Noorahya Jambari and Farina Mustaffa-Kamal

Molecules 2020, 25(11), 2622; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25112622 - 05 Jun 2020

Cited by 7 | Viewed by 3226

Abstract

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of α_s1-casein. Detection and quantification of α_s1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat α_s1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat α_s1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat α_s1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat α_s1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat α_s1-casein, with lower K_d value at 5.063 × 10⁻³ μM compared to 9.046 × 10⁻³ μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat α_s1-casein. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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18 pages, 2586 KiB

Open AccessArticle

A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett–Burman and Box–Behnken Designs

by Sorel Tchewonpi Sagu, Gerd Huschek, Josephine Bönick, Thomas Homann and Harshadrai M. Rawel

Molecules 2019, 24(19), 3589; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24193589 - 05 Oct 2019

Cited by 21 | Viewed by 4061

Abstract

Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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11 pages, 1880 KiB

Open AccessArticle

Purification and Identification of Antioxidant Peptides from Schizochytrium Limacinum Hydrolysates by Consecutive Chromatography and Electrospray Ionization-Mass Spectrometry

by Xiao Hu, Xianqing Yang, Qiong Wu, Laihao Li, Yanyan Wu, Shengjun Chen, Ruijie Li and Jiaoyan Ren

Molecules 2019, 24(16), 3004; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules24163004 - 19 Aug 2019

Cited by 16 | Viewed by 3194

Abstract

Schizochytrium limacinum residue was hydrolyzed with various proteases (papain, trypsin, Flavourzyme, Protamex, and Alcalase 2.4L) to obtain antioxidative peptides. The results showed that the S. limacinum hydrolysates (SLHs) prepared with compound proteases (Protamex and Alcalase 2.4L) had the highest antioxidant activity, which was measured using methods such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability (IC₅₀ = 1.28 mg/mL), hydroxyl radical scavenging ability (IC₅₀ = 1.66 mg/mL), and reducing power (1.42 at 5.0 mg/mL). The hydrolysates were isolated and purified by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of electrospray ionization-mass spectrometer (ESI-MS/MS), the purified antioxidant peptide was identified as Pro-Tyr-Lys (406 Da). Finally, the identified peptide was synthesized for evaluating its antioxidant activity. The •OH scavenging ability and reducing power of Pro-Tyr-Lys were comparable to those of reduced L-glutathione (GSH). These results demonstrated that the antioxidant peptides from SLHs could potentially be used as effective antioxidants. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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Review

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17 pages, 14683 KiB

Open AccessFeature PaperReview

Methods of Purification and Application Procedures of Alpha1 Antitrypsin: A Long-Lasting History

by Simona Viglio, Paolo Iadarola, Maura D’Amato and Jan Stolk

Molecules 2020, 25(17), 4014; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules25174014 - 02 Sep 2020

Cited by 7 | Viewed by 8680

Abstract

The aim of the present report is to review the literature addressing the methods developed for the purification of alpha1-antitrypsin (AAT) from the 1950s to the present. AAT is a glycoprotein whose main function is to protect tissues from human neutrophil elastase (HNE) and other proteases released by neutrophils during an inflammatory state. The lack of this inhibitor in human serum is responsible for the onset of alpha1-antitrypsin deficiency (AATD), which is a severe genetic disorder that affects lungs in adults and for which there is currently no cure. Being used, under special circumstances, as a medical treatment of AATD in the so-called “replacement” therapy (consisting in the intravenous infusion of the missing protein), AAT is a molecule with a lot of therapeutic importance. For this reason, interest in AAT purification from human plasma or its production in a recombinant version has grown considerably in recent years. This article retraces all technological advances that allowed the manufacturers to move from a few micrograms of partially purified AAT to several grams of highly purified protein. Moreover, the chronic augmentation and maintenance therapy in individuals with emphysema due to congenital AAT deficiency (current applications in the clinical setting) is also presented. Full article

(This article belongs to the Special Issue The Optimized Production, Purification, Characterization and Application of Proteins/Enzymes)

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