scholarly journals ‘Traceless’ tracing of proteins – high-affinity trans-splicing directed by a minimal interaction pair

2016 ◽  
Vol 7 (4) ◽  
pp. 2646-2652 ◽  
Author(s):  
M. Braner ◽  
A. Kollmannsperger ◽  
R. Wieneke ◽  
R. Tampé
Keyword(s):  
Mammalian Cells ◽  
Protein Labeling ◽  
Terminal Protein ◽  
Site Specific ◽  
High Affinity ◽  
Trans Splicing

Using a minimal lock-and-key element the affinity between the intein fragments for N-terminal protein trans-splicing was significantly increased, allowing for site-specific, ‘traceless’ covalent protein labeling in living mammalian cells at nanomolar probe concentrations.

Nanomolar affinity protein trans-splicing monitored in real-time by fluorophore–quencher pairs

2017 ◽  
Vol 53 (3) ◽  
pp. 545-548 ◽  
Author(s):  
M. Braner ◽  
R. Wieneke ◽  
R. Tampé
Keyword(s):  
Real Time ◽  
Protein Labeling ◽  
Online Detection ◽  
Physiological Conditions ◽  
High Affinity ◽  
Cellular Environment ◽  
Trans Splicing

We combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal ‘traceless’ protein labeling at nanomolar concentrations under physiological conditions in cellular environment.

Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells

2016 ◽  
Vol 15 (3) ◽  
pp. 1103-1113 ◽  
Author(s):  
Tong Zhou ◽  
Ying-hua Chung ◽  
Jianji Chen ◽  
Yue Chen
Keyword(s):  
Mammalian Cells ◽  
Lysine Acetylation ◽  
Site Specific ◽  
Specific Identification

scholarly journals Use of an Asparaginyl Endopeptidase for Chemo-enzymatic Peptide and Protein Labeling

2020 ◽  
Author(s):  
Simon Tang ◽  
Davide Cardella ◽  
Alexander J. Lander ◽  
Xuefei Li ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Phenylboronic Acid ◽  
Product Formation ◽  
Protein Labeling ◽  
Recognition Sequence ◽  
Reaction Conditions ◽  
Site Specific ◽  
Asparaginyl Endopeptidase ◽  
Coupling System ◽  
Labeling System ◽  
Specific Peptide

Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.

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