scholarly journals Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins

2021 ◽  
Vol 22 (8) ◽  
pp. 4058
Author(s):  
Lukas Pfeifer ◽  
Alexander Baumann ◽  
Lea Madlen Petersen ◽  
Bastian Höger ◽  
Eric Beitz ◽  
...  
Keyword(s):  
Zostera Marina ◽  
Echinacea Purpurea ◽  
Arabinogalactan Proteins ◽  
Uronic Acids ◽  
Binding Properties ◽  
Free System ◽  
Plant Polysaccharides ◽  
Kd Values ◽  
A Cell ◽  
Partial Degradation

Galectins represent β-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1–2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08–0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

scholarly journals Effect of detergents on sterol synthesis in a cell-free system of yeast

1982 ◽  
Vol 23 (6) ◽  
pp. 803-810
Author(s):  
S Hata ◽  
T Nishino ◽  
N Ariga ◽  
H Katsuki
Keyword(s):  
Sterol Synthesis ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of Endosomal Proteolysis in a Cell-free System

1989 ◽  
Vol 264 (10) ◽  
pp. 5392-5399
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
P D Stahl
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Anne Stinn ◽  
Jens Furkert ◽  
Stefan H. E. Kaufmann ◽  
Pedro Moura-Alves ◽  
Michael Kolbe
Keyword(s):  
Ligand Binding ◽  
Environmental Pollutants ◽  
Screening Strategy ◽  
Pharmacological Intervention ◽  
Binding Affinities ◽  
Free System ◽  
Microscale Thermophoresis ◽  
Aryl Hydrocarbon ◽  
Promising Target ◽  
A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

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