scholarly journals Identification of a Recombinant Human Interleukin-12 (rhIL-12) Fragment in Non-Reduced SDS-PAGE

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1210 ◽  
Author(s):  
Lei Yu ◽  
Yonghong Li ◽  
Lei Tao ◽  
Chuncui Jia ◽  
Wenrong Yao ◽  
...  
Keyword(s):  
Quality Control ◽  
Size Exclusion ◽  
Interleukin 12 ◽  
Small Fragment ◽  
Preclinical Models ◽  
Critical Quality Attribute ◽  
Native Page ◽  
The Past ◽  
Sds Page ◽  
Exclusion Chromatography

During the past two decades, recombinant human interleukin-12 (rhIL-12) has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models and clinical studies. Purity is a critical quality attribute (CQA) in the quality control system of rhIL-12. In our study, rhIL-12 bulks from manufacturer B showed a different pattern in non-reduced SDS-PAGE compared with size-exclusion chromatography (SEC)-HPLC. A small fragment was only detected in non-reduced SDS-PAGE but not in SEC-HPLC. The results of UPLC/MS and N-terminal sequencing confirmed that the small fragment was a 261–306 amino acid sequence of a p40 subunit of IL-12. The cleavage occurs between Lys260 and Arg261, a basic rich region. With the presence of 0.2% SDS, the small fragment appeared in both native PAGE and in SEC-HPLC, suggesting that it is bound to the remaining part of the IL-12 non-covalently, and is dissociated in a denatured environment. The results of a bioassay showed that the fractured rhIL-12 proteins had deficient biological activity. These findings provide an important reference for the quality control of the production process and the final products of rhIL-12.

scholarly journals Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

2005 ◽  
Vol 48 (5) ◽  
pp. 705-716 ◽  
Author(s):  
Maria Aparecida Souza ◽  
Francielle Amâncio-Pereira ◽  
Cristina Ribeiro Barros Cardoso ◽  
Adriano Gomes da Silva ◽  
Edmar Gomes Silva ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Terminal Sequence ◽  
Native Page ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Polypeptide Chains ◽  
Binding Lectin

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

scholarly journals The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

2002 ◽  
Vol 13 (11) ◽  
pp. 3811-3821 ◽  
Author(s):  
Pauli J. Ojala ◽  
Ville O. Paavilainen ◽  
Maria K. Vartiainen ◽  
Roman Tuma ◽  
Alan G. Weeds ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Activity ◽  
Size Exclusion ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Monomer ◽  
Wild Type ◽  
Native Page ◽  
Exclusion Chromatography

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K D = 0.05 μM) than ATP-G-actin (K D = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k off = 1.8 s−1) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2455-2462 ◽  
Author(s):  
Masaru Nagai ◽  
Maki Kawata ◽  
Hisayuki Watanabe ◽  
Machiko Ogawa ◽  
Kumiko Saito ◽  
...  
Keyword(s):  
Lentinula Edodes ◽  
Veratryl Alcohol ◽  
Size Exclusion ◽  
Melanin Synthesis ◽  
Sds Page ◽  
Diammonium Salt ◽  
Exclusion Chromatography ◽  
Homogeneous Preparation

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

scholarly journals The rapid “teabag” method for high-end purification of membrane proteins

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jenny Hering ◽  
Julie Winkel Missel ◽  
Liying Zhang ◽  
Anders Gunnarsson ◽  
Marie Castaldo ◽  
...  
Keyword(s):  
Applied Sciences ◽  
Membrane Proteins ◽  
Size Exclusion ◽  
Purification Method ◽  
New Approach ◽  
Purification Time ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Processing Steps

Abstract Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast “teabag” purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.

Size exclusion chromatography for the characterization and quality control of biologics

2021 ◽  
pp. 1-14
Author(s):  
Surinder M. Singh ◽  
Ran Furman ◽  
Rajesh K. Singh ◽  
Gurusamy Balakrishnan ◽  
Naresh Chennamsetty ◽  
...  
Keyword(s):  
Quality Control ◽  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Markedly Increased Vitamin B12 Concentrations Attributable to IgG–IgM–Vitamin B12 Immune Complexes

2006 ◽  
Vol 52 (11) ◽  
pp. 2107-2114 ◽  
Author(s):  
Raffick AR Bowen ◽  
Steven K Drake ◽  
Rachna Vanjani ◽  
Edward D Huey ◽  
Jordan Grafman ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Immune Complexes ◽  
Size Exclusion ◽  
Protein G ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Tof Ms

Abstract Background: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient’s serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient’s serum revealed an abnormal vitamin-B12-binding protein. SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient’s eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

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