Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

10.3791/61918 ◽  
2021 ◽  
Author(s):  
Mateusz Adam Krzyscik ◽  
Aleksandra Sokolowska-Wedzina ◽  
Karolina Jendryczko ◽  
Marta Pozniak ◽  
Daria Nawrocka ◽  
...  
Keyword(s):  
Sortase A ◽  
Site Specific ◽  
Protein Conjugates ◽  
Cytotoxic Protein

scholarly journals Bioorthogonal protein-DNA conjugation methods for force spectroscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Synakewicz ◽  
Daniela Bauer ◽  
Matthias Rief ◽  
Laura S. Itzhaki
Keyword(s):  
Single Molecule ◽  
Unnatural Amino Acids ◽  
Force Spectroscopy ◽  
Diels Alder ◽  
Cysteine Residues ◽  
Single Molecule Force Spectroscopy ◽  
Site Specific ◽  
Inverse Electron Demand ◽  
Protein Conjugates ◽  
Electron Demand

Abstract Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible.

Site-specific polymer–protein conjugates by Cys mutation

2020 ◽  
pp. 297-316
Author(s):  
Nandini V. Katre
Keyword(s):  
Site Specific ◽  
Protein Conjugates

scholarly journals Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool

Soft Matter ◽  
2013 ◽  
Vol 9 (29) ◽  
pp. 6752 ◽  
Author(s):  
Susanna Piluso ◽  
Heather C. Cassell ◽  
Jonathan L. Gibbons ◽  
Thomas E. Waller ◽  
Nick J. Plant ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Sortase A ◽  
Site Specific ◽  
Self Assembling ◽  
Peptide Hydrogels

Site-specific tetrameric streptavidin-protein conjugation using sortase A

2011 ◽  
Vol 152 (1-2) ◽  
pp. 37-42 ◽  
Author(s):  
Takuya Matsumoto ◽  
Shiori Sawamoto ◽  
Takayuki Sakamoto ◽  
Tsutomu Tanaka ◽  
Hideki Fukuda ◽  
...  
Keyword(s):  
Sortase A ◽  
Protein Conjugation ◽  
Site Specific

scholarly journals Three-in-One Chromatography-Free Purification, Tag Removal, and Site-Specific Modification of Recombinant Fusion Proteins Using Sortase A and Elastin-like Polypeptides

2013 ◽  
Vol 52 (13) ◽  
pp. 3703-3708 ◽  
Author(s):  
Joseph J. Bellucci ◽  
Miriam Amiram ◽  
Jayanta Bhattacharyya ◽  
Dewey McCafferty ◽  
Ashutosh Chilkoti
Keyword(s):  
Fusion Proteins ◽  
Sortase A ◽  
Site Specific ◽  
Specific Modification ◽  
Recombinant Fusion Proteins

scholarly journals Capture and Recycling of Sortase A through Site-Specific Labeling with Lithocholic Acid

2016 ◽  
Vol 55 (30) ◽  
pp. 8585-8589 ◽  
Author(s):  
Christian B. Rosen ◽  
Richard L. Kwant ◽  
James I. MacDonald ◽  
Meera Rao ◽  
Matthew B. Francis
Keyword(s):  
Lithocholic Acid ◽  
Sortase A ◽  
Site Specific

Dual Site-Specific Labeling of an Antibody Fragment through Sortase A and π-Clamp Conjugation

2019 ◽  
Vol 30 (10) ◽  
pp. 2539-2543 ◽  
Author(s):  
Michael D. Lee ◽  
Wing Yin Tong ◽  
Thomas Nebl ◽  
Lesley A. Pearce ◽  
Tam M. Pham ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Sortase A ◽  
Site Specific ◽  
Dual Site

scholarly journals New Method for Site-Specific Modification of Liposomes with Proteins Using Sortase A-Mediated Transpeptidation

2012 ◽  
Vol 23 (3) ◽  
pp. 650-655 ◽  
Author(s):  
Xueqing Guo ◽  
Zhimeng Wu ◽  
Zhongwu Guo
Keyword(s):  
New Method ◽  
Sortase A ◽  
Site Specific ◽  
Specific Modification
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