Methods for Characterization of Protein Aggregates

2014 ◽  
pp. 387-401 ◽  
Author(s):  
Witold Tatkiewicz ◽  
Elisa Elizondo ◽  
Evelyn Moreno ◽  
Cesar Díez-Gil ◽  
Nora Ventosa ◽  
...  
Keyword(s):  
Protein Aggregates

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach

2019 ◽  
Vol 25 (1) ◽  
pp. 164-179
Author(s):  
Ambroise Marin ◽  
Emmanuel Denimal ◽  
Lucie Bertheau ◽  
Stéphane Guyot ◽  
Ludovic Journaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Protein Aggregates ◽  
Point Of View ◽  
Zernike Moment ◽  
Two Photon ◽  
Haralick Features ◽  
Green Fluorescent ◽  
Two Photon Fluorescence

AbstractIn the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics

2015 ◽  
Vol 67 (2) ◽  
pp. 616-624 ◽  
Author(s):  
Adriana Bajardi-Taccioli ◽  
Andrew Blum ◽  
Chongfeng Xu ◽  
Zoran Sosic ◽  
Svetlana Bergelson ◽  
...  
Keyword(s):  
Protein Aggregates

Characterization of the fragmentation of protein aggregates in suspension subjected to flow

2007 ◽  
Vol 62 (22) ◽  
pp. 6440-6450 ◽  
Author(s):  
N. Péron ◽  
S.P. Heffernan ◽  
E.P. Byrne ◽  
F. Rioual ◽  
J.J. Fitzpatrick
Keyword(s):  
Protein Aggregates

scholarly journals Evaluation of nanoparticle tracking for characterization of fibrillar protein aggregates

AIChE Journal ◽  
2014 ◽  
Vol 60 (4) ◽  
pp. 1236-1244 ◽  
Author(s):  
Dennis T. Yang ◽  
Xiaomeng Lu ◽  
Yamin Fan ◽  
Regina M. Murphy
Keyword(s):  
Protein Aggregates

scholarly journals Aggregation-prone peptides modulate interferon gamma functionality in naturally occurring protein nanoparticles

2019 ◽  
Author(s):  
José Vicente Carratalá ◽  
Olivia Cano-Garrido ◽  
Julieta Sánchez ◽  
Cristina Membrado ◽  
Eudald Pérez ◽  
...  
Keyword(s):  
Biological Activity ◽  
Protein Aggregation ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Expression System ◽  
Protein Aggregates ◽  
Naturally Occurring ◽  
Protein Nanoparticles ◽  
Ifn Γ

AbstractEfficient protocols for the production of recombinant proteins are indispensable for the development of the biopharmaceutical sector. Approximately 400 recombinant protein-based biopharmaceuticals have been approved in recent decades, with steady growth projected in the coming years. During the expression of a heterologous gene, the protein quality control network is overcome by the disruption in protein homeostasis, leading to protein aggregation. This phenomenon has been described in all expression systems analyzed to date, including prokaryotic and eukaryotic host cells. These protein aggregates have long been considered inert protein clumps devoid of biological activity and their study has largely been neglected. However, in recent years, the classic view of protein aggregates has completely changed with the recognition that these aggregates are a valuable source of functional recombinant proteins. In this study, bovine interferon-gamma (rBoIFN-γ) was engineered to enhance the formation of protein aggregates by the addition of aggregation-prone peptides (APPs) in the generally recognized as safe (GRAS) bacterial Lactococcus lactis expression system. The L6K2, HALRU and CYOB peptides were selected to assess their intrinsic aggregation capability to nucleate protein aggregation. These APPs enhanced the tendency of the resulting protein to aggregate at the expense of the total protein yield. However, fine physicochemical characterization of the resulting intracellular protein nanoparticles (NPs), the protein released from these protein NPs, and the protein purified from the soluble cell fraction indicated that the compactability of protein conformations is directly related to the biological activity of variants of IFN-γ, which is used here as a model protein with therapeutic potential.ImportanceThe demand for recombinant proteins in the pharmaceutical industry is steadily increasing. Emerging novel protein formulations, including naturally occurring protein NPs, might be an alternative to soluble variants for fine analysis at the biophysical level. Such analyses are important to address safety about biological molecules.This study analyzes the effect of aggregation-prone peptides (APPs) on the improvement of the production of naturally occurring protein nanoparticles (NPs) of interferon gamma (IFN-γ) in the generally recognized as safe (GRAS) Lactococcus lactis expression system. In addition, the fine physico-chemical characterization of the resulting proteins, either obtained from the soluble or insoluble cell fractions, indicates that the selected engineered proteins embedded in the protein NPs show higher compactability than their soluble protein counterparts. Conformational compactability is directly related to the biological performance of the recombinant IFN-γ.

Size-selective concentration and label-free characterization of protein aggregates using a Raman active nanofluidic device

Lab on a Chip ◽  
2011 ◽  
Vol 11 (4) ◽  
pp. 632-638 ◽  
Author(s):  
Inhee Choi ◽  
Yun Suk Huh ◽  
David Erickson
Keyword(s):  
Protein Aggregates ◽  
Label Free ◽  
Nanofluidic Device

scholarly journals In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Diletta Ami ◽  
Francesca Lavatelli ◽  
Paola Rognoni ◽  
Giovanni Palladini ◽  
Sara Raimondi ◽  
...  
Keyword(s):  
Light Chain ◽  
Protein Aggregates ◽  
Human Tissues ◽  
In Situ Characterization ◽  
Light Chain Amyloidosis ◽  
Ftir Microspectroscopy

Selective autophagy

2013 ◽  
Vol 55 ◽  
pp. 79-92 ◽  
Author(s):  
Steingrim Svenning ◽  
Terje Johansen
Keyword(s):  
Light Chain ◽  
Bacterial Pathogens ◽  
Protein Aggregates ◽  
Degradation Pathway ◽  
Intracellular Bacteria ◽  
Related Protein ◽  
Lysosomal Degradation ◽  
Selective Autophagy

During the last decade it has become evident that autophagy is not simply a non-selective bulk degradation pathway for intracellular components. On the contrary, the discovery and characterization of autophagy receptors which target specific cargo for lysosomal degradation by interaction with ATG8 (autophagy-related protein 8)/LC3 (light-chain 3) has accelerated our understanding of selective autophagy. A number of autophagy receptors have been identified which specifically mediate the selective autophagosomal degradation of a variety of cargoes including protein aggregates, signalling complexes, midbody rings, mitochondria and bacterial pathogens. In the present chapter, we discuss these autophagy receptors, their binding to ATG8/LC3 proteins and how they act in ubiquitin-mediated selective autophagy of intracellular bacteria (xenophagy) and protein aggregates (aggrephagy).

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