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22 pages, 4066 KiB

Open AccessArticle

Identification and Characterization of Novel Fc-Binding Heptapeptides from Experiments and Simulations

by Xiaoquan Sun, Justin Weaver, Sumith Ranil Wickramasinghe and Xianghong Qian

Polymers 2018, 10(7), 778; https://doi.org/10.3390/polym10070778 - 16 Jul 2018

Cited by 5 | Viewed by 3956

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products, therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity [...] Read more.

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products, therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 phage display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations. Full article

(This article belongs to the Special Issue Polymers for Bioseparations)

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12 pages, 1881 KiB

Open AccessArticle

Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags

by Yamin Fan, Jackelyn M. Miozzi, Samuel D. Stimple, Tzu-Chiang Han and David W. Wood

Polymers 2018, 10(5), 468; https://doi.org/10.3390/polym10050468 - 25 Apr 2018

Cited by 29 | Viewed by 6691

Abstract

Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant [...] Read more.

Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant drawback, however, is that current proteolytic approaches for post-purification tag removal after are expensive and non-scalable. To address this problem, we have developed two non-chromatographic purification strategies that use either the elastin-like polypeptide (ELP) tag or the β-roll tag (BRT17) in combination with an engineered split intein for tag removal. The use of the split intein eliminates premature cleavage during expression and provides controlled cleavage under mild conditions after purification. These self-cleaving aggregating tags were used to efficiently purify β-lactamase (β-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), resulting in increased yields compared to previous ELP and BRT17-based methods. Observed yields of purified targets for both systems typically ranged from approximately 200 to 300 micrograms per milliliter of cell culture, while overall recoveries ranged from 10 to 85 percent and were highly dependent on the target protein. Full article

(This article belongs to the Special Issue Polymers for Bioseparations)

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15 pages, 1361 KiB

Open AccessArticle

Statistical Design of Experimental and Bootstrap Neural Network Modelling Approach for Thermoseparating Aqueous Two-Phase Extraction of Polyhydroxyalkanoates

by Yoong Kit Leong, Chih-Kai Chang, Senthil Kumar Arumugasamy, John Chi-Wei Lan, Hwei-San Loh, Dinie Muhammad and Pau Loke Show

Polymers 2018, 10(2), 132; https://doi.org/10.3390/polym10020132 - 30 Jan 2018

Cited by 7 | Viewed by 4626

Abstract

At present, polyhydroxyalkanoates (PHAs) have been considered as a promising alternative to conventional plastics due to their diverse variability in structure and rapid biodegradation. To ensure cost competitiveness in the market, thermoseparating aqueous two-phase extraction (ATPE) with the advantages of being mild and [...] Read more.

At present, polyhydroxyalkanoates (PHAs) have been considered as a promising alternative to conventional plastics due to their diverse variability in structure and rapid biodegradation. To ensure cost competitiveness in the market, thermoseparating aqueous two-phase extraction (ATPE) with the advantages of being mild and environmental-friendly was suggested as the primary isolation and purification tool for PHAs. Utilizing two-level full factorial design, this work studied the influence and interaction between four independent variables on the partitioning behavior of PHAs. Based on the experimental results, feed forward neural network (FFNN) was used to develop an empirical model of PHAs based on the ATPE thermoseparating input-output parameter. In this case, bootstrap resampling technique was used to generate more data. At the conditions of 15 wt % phosphate salt, 18 wt % ethylene oxide–propylene oxide (EOPO), and pH 10 without the addition of NaCl, the purification and recovery of PHAs achieved a highest yield of 93.9%. Overall, the statistical analysis demonstrated that the phosphate concentration and thermoseparating polymer concentration were the most significant parameters due to their individual influence and synergistic interaction between them on all the response variables. The final results of the FFNN model showed the ability of the model to seamlessly generalize the relationship between the input–output of the process. Full article

(This article belongs to the Special Issue Polymers for Bioseparations)

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