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

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 33950

Special Issue Editor

Prof. Dr. Paolo Iadarola

E-Mail Website
Guest Editor
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
Special Issues, Collections and Topics in MDPI journals

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

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Published Papers (7 papers)

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Research

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14 pages, 4260 KiB  
Article
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://doi.org/10.3390/molecules26051258 - 26 Feb 2021
Cited by 2 | Viewed by 2479
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 [...] Read more.
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  
Article
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://doi.org/10.3390/molecules25184071 - 6 Sep 2020
Cited by 10 | Viewed by 3518
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 [...] Read more.
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  
Article
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://doi.org/10.3390/molecules25112625 - 5 Jun 2020
Cited by 10 | Viewed by 4468
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 [...] Read more.
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  
Article
Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-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://doi.org/10.3390/molecules25112622 - 5 Jun 2020
Cited by 7 | Viewed by 3865
Abstract
The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability [...] Read more.
The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-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 αs1-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 αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-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 αs1-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 αs1-casein, with lower Kd 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 αs1-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  
Article
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://doi.org/10.3390/molecules24193589 - 5 Oct 2019
Cited by 23 | Viewed by 4676
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 [...] Read more.
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  
Article
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://doi.org/10.3390/molecules24163004 - 19 Aug 2019
Cited by 19 | Viewed by 3624
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 [...] Read more.
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  
Review
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://doi.org/10.3390/molecules25174014 - 2 Sep 2020
Cited by 9 | Viewed by 10035
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) [...] Read more.
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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