scholarly journals Expression of Monomeric Insulin Precursor in Pichia pastoris and Its Conversion into Monomeric B27 Lys Destripeptide Insulin by Tryptic Hydrolysis

2005 ◽  
Vol 37 (4) ◽  
pp. 234-240 ◽  
Author(s):  
Jin-Guo Ding ◽  
Jian Fei ◽  
Da-Fu Cui ◽  
You-Shang Zhang
Keyword(s):  
Pichia Pastoris ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Exclusion Chromatography ◽  
Native Gel ◽  
Tryptic Hydrolysis

Abstract Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition, and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.

scholarly journals Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

2018 ◽  
Author(s):  
Bingyu Ye ◽  
Wenlong Shen ◽  
Minglei Shi ◽  
Yan Zhang ◽  
Cunshuan Xu ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Clinical Investigation ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Radioprotective Activity ◽  
Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

scholarly journals Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5043
Author(s):  
Bingyu Ye ◽  
Wenlong Shen ◽  
Minglei Shi ◽  
Yan Zhang ◽  
Cunshuan Xu ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Clinical Investigation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Nostoc Punctiforme ◽  
Radioprotective Activity ◽  
Backbone Cyclization

Background Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods We designed and constructed cyclic entolimod using split Nostoc punctiforme DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Both of linear and circular entolimod were first purified by Ni-chelating affinity chromatography, and then the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Results The circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Conclusion Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

scholarly journals Intein-mediated backbone cyclization of entolimod confers enhanced radioprotective activity in mouse models

2018 ◽  
Author(s):  
Bingyu Ye ◽  
Wenlong Shen ◽  
Minglei Shi ◽  
Yan Zhang ◽  
Cunshuan Xu ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Clinical Investigation ◽  
Affinity Purification ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Radioprotective Activity ◽  
Backbone Cyclization

Background. Entolimod is a Salmonella enterica flagellin derivate. Previous work has demonstrated that entolimod effectively protects mice and non-human primates from ionizing radiation. However, it caused a “flu-like” syndrome after radioprotective and anticancer clinical application, indicating some type of immunogenicity and toxicity. Cyclization is commonly used to improve the in vivo stability and activity of peptides and proteins. Methods. We designed and constructed cyclic entolimod using split Npu DnaE intein with almost 100% cyclization efficiency. We adopted different strategies to purify the linear and circular entolimod due to their different topologies. Results. After Ni-chelating affinity purification, the linear and circular entolimod were purified by size-exclusion and ion-exchange chromatography, respectively. Compared with linear entolimod, the circular entolimod showed significantly increased both the in vitro NF-κB signaling and in vivo radioprotective activity in mice. Discussions/Conclusions. Our data indicates that circular entolimod might be a good candidate for further clinical investigation.

scholarly journals Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis

2016 ◽  
Vol 63 (3) ◽  
Author(s):  
Marcin Grąz ◽  
Kamila Rachwał ◽  
Radosław Zan ◽  
Anna Jarosz-Wilkołazka
Keyword(s):  
Oxalic Acid ◽  
Ph Value ◽  
Enzyme Reaction ◽  
Oxalate Oxidase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Optimum Temperature ◽  
Intracellular Enzyme ◽  
Exclusion Chromatography

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min-1, respectively.

scholarly journals Structure of the omalizumab Fab

2015 ◽  
Vol 71 (4) ◽  
pp. 419-426 ◽  
Author(s):  
Rasmus K. Jensen ◽  
Melanie Plum ◽  
Luna Tjerrild ◽  
Thilo Jakob ◽  
Edzard Spillner ◽  
...  
Keyword(s):  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Structural Basis ◽  
Hek 293 ◽  
Functional Studies ◽  
293 Cells ◽  
Exclusion Chromatography ◽  
Ige Mediated ◽  
High Affinity Ige Receptor

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

Abstract 255: Plasminogen Promotes Cholesterol Efflux by the ABCA1

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Nathalie Pamir ◽  
David A Dichek ◽  
Godfrey S Getz ◽  
Santica Marcovina ◽  
Jay W Heinecke
Keyword(s):  
Size Exclusion Chromatography ◽  
Cholesterol Efflux ◽  
Particle Concentration ◽  
Specific Activity ◽  
Cholesterol Homeostasis ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Cvd Risk ◽  
Exclusion Chromatography

Background: The cholesterol efflux capacity (CEC) of serum HDL, measured using cultured macrophages predicts incident and prevalent CVD risk in humans. The ABCA1 pathway is a key regulator of macrophage cholesterol homeostasis in vivo. Methods: We used genetic and biochemical approaches in mice to identify important mediators of CEC. Results: On high-resolution size-exclusion chromatography of mouse plasma, macrophage CEC and HDL co-eluted as a single major peak, suggesting that HDL mediates cholesterol efflux. In contrast, size-exclusion chromatography revealed two major peaks of material that promoted ABCA1-specific CEC, one of which was distinct from HDL. HDL particle concentration was reduced by 75% in Apoa1 -/- mice; this resulted in a 50% decrease in macrophage CEC but, surprisingly, had no impact on ABCA1-specific CEC. Orthogonal chromatography-mass spectrometric analysis of the non-HDL-associated efflux inducing material isolated from wild-type and APOA1 deficient plasma showed that plasminogen strongly correlated with ABCA1-specific CEC. Moreover, isolated plasminogen promoted cholesterol efflux by the ABCA1 pathway, and the specific activity of ABCA1-specific CEC of non-HDL-associated material was reduced by 50% in plasminogen deficient plasma. Imaging of cells treated with fluorescently-labeled antibodies demonstrated that ABCA1 and plasminogen co-localized on the plasma membrane. Conclusions: HDL particle concentration is an important contributor to macrophage CEC. However, other pathways contribute to ABCA1-specific CEC; our studies identify plasminogen as one potential mediator. Plasminogen associates with CVD risk in human genetic studies, raising the possibility that it plays a role in atherosclerosis by modulating ABCA1-mediated sterol efflux from macrophages.

Large-scale preparation of α-D-(14)-oligogalacturonic acids from pectic acid

2002 ◽  
Vol 80 (8) ◽  
pp. 900-903 ◽  
Author(s):  
Hong-Ni Fan ◽  
Mei-Zheng Liu ◽  
Yuan C Lee
Keyword(s):  
Galacturonic Acid ◽  
Large Scale ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Inexpensive Method ◽  
Pectic Acid ◽  
Large Scale Preparation ◽  
Exclusion Chromatography ◽  
Scale Preparation

An efficient and inexpensive method for large-scale preparation of α-D-(1[Formula: see text]4)-oligogalacturonic acids (oligo-GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure – size-exclusion chromatography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2-5. Key words: pectic acid, galacturonic acid, galabiose, galatriose, pectinase.

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