scholarly journals A designed repeat protein as an affinity capture reagent

2015 ◽  
Vol 43 (5) ◽  
pp. 874-880 ◽  
Author(s):  
Elizabeth B. Speltz ◽  
Rebecca S.H. Brown ◽  
Holly S. Hajare ◽  
Christian Schlieker ◽  
Lynne Regan
Keyword(s):  
Affinity Purification ◽  
Basic Research ◽  
Tetratricopeptide Repeat ◽  
Affinity Capture ◽  
Practical Applications ◽  
General Utility ◽  
Repeat Proteins ◽  
Peptide Interactions ◽  
Peptide Interaction ◽  
Acta Crystallogr

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class: tetratricopeptide repeat (TPR) proteins. In previous work, we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena (2011) Prot. Sci. 20, 1042–1047; Main (2003) Structure 11, 497–508]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena (2009) ACS Chem. Biol. 5, 545—552; Cortajarena (2008) ACS Chem. Biol. 3, 161—166; Jackrel (2009) Prot. Sci. 18, 762—774; Kajander (2007) Acta Crystallogr. D Biol. Crystallogr. 63, 800—811]. Here we focus on the development of one such TPR–peptide interaction for a practical application, affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein–peptide interactions can lead to the development of novel reagents with important practical applications.

Quantum Dots in basic research and practical applications: the role of size and quasi-multivalency

2018 ◽  
Author(s):  
A.V. Salova ◽  
T.N. Belyaeva ◽  
V.V. Kosheverova ◽  
E.A. Leontieva ◽  
M.V. Kharchenko ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Basic Research ◽  
Practical Applications

Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi

2016 ◽  
Vol 54 (2) ◽  
pp. 136-144 ◽  
Author(s):  
Sunyoung Bang ◽  
Chan-Ki Min ◽  
Na-Young Ha ◽  
Myung-Sik Choi ◽  
Ik-Sang Kim ◽  
...  
Keyword(s):  
Tetratricopeptide Repeat ◽  
Orientia Tsutsugamushi ◽  
Eukaryotic Translation ◽  
Repeat Proteins ◽  
Tetratricopeptide Repeat Proteins

scholarly journals Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1070 ◽  
Author(s):  
Danielle G. May ◽  
Kelsey L. Scott ◽  
Alexandre R. Campos ◽  
Kyle J. Roux
Keyword(s):  
Protein Interactions ◽  
Culture Media ◽  
Affinity Purification ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Experimental Protocol ◽  
Established Method ◽  
Cellular Toxicity ◽  
Practical Applications ◽  
Protein Instability

BioID is a well-established method for identifying protein–protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15–18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

scholarly journals A Molecular View of Autophagy in Lepidoptera

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Davide Romanelli ◽  
Barbara Casati ◽  
Eleonora Franzetti ◽  
Gianluca Tettamanti
Keyword(s):  
Basic Research ◽  
Larval Body ◽  
New Era ◽  
Practical Applications ◽  
The Past ◽  
Insect Order ◽  
Lepidopteran Species ◽  
Adult Insect ◽  
Critical Phase

Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm,Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications.

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