Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase

PloS One
Samuel K Oteng-PabiJeffrey W Keillor

Abstract

Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences wer...Continue Reading

References

Jul 1, 1997·Bioscience, Biotechnology, and Biochemistry·Y KumazawaK Seguro
Oct 21, 1999·Analytical Biochemistry·N Day, J W Keillor
Jan 24, 2004·Applied Microbiology and Biotechnology·K YokoyamaY Kikuchi
Feb 3, 2004·Journal of Receptor and Signal Transduction Research·Michael Szardenings
Aug 20, 2004·Journal of Bacteriology·Katerina Ragkousi, Peter Setlow
Nov 18, 2005·Analytical Biochemistry·Steve M F G GilletJeffrey W Keillor
Dec 3, 2005·Journal of Bacteriology·D C McPhersonA Driks
Nov 10, 2006·Bioorganic & Medicinal Chemistry Letters·Dany HalimJeffrey W Keillor
Aug 19, 2008·Trends in Biotechnology·Yang Zhu, Johannes Tramper
Jan 22, 2010·Journal of Agricultural and Food Chemistry·Kingsley K Agyare, Srinivasan Damodaran
Jun 11, 2010·Proceedings of the National Academy of Sciences of the United States of America·Chayasith UttamapinantAlice Y Ting
Feb 1, 2011·Cellular & Molecular Immunology·Sergio CajaKatri Lindfors
Jan 1, 2010·Biotechnology & Genetic Engineering Reviews·Dongxu ZhangJian Chen
Oct 27, 2011·Molecular Microbiology·Thomas SpirigRobert T Clubb
Aug 14, 2012·Biotechnology and Bioengineering·Jae-Hun LeeByung-Gee Kim
Apr 26, 2013·Applied Microbiology and Biotechnology·Maria T GundersenJoelle N Pelletier
Jun 7, 2013·Biotechnology and Bioengineering·Jae-Hun LeeByung-Gee Kim
Jul 11, 2013·Bioconjugate Chemistry·Mohammad RashidianMark D Distefano
Jul 24, 2013·Analytical Biochemistry·Samuel K Oteng-Pabi, Jeffrey W Keillor
Nov 8, 2013·Folia Microbiologica·Marek Kieliszek, Anna Misiewicz
Apr 4, 2014·Bioconjugate Chemistry·Pavel Strop
May 16, 2014·Chemical Communications : Chem Comm·Samuel K Oteng-PabiJeffrey W Keillor
Jun 18, 2014·Nature Chemical Biology·Kevin M Dean, Amy E Palmer
Jun 21, 2014·Journal of Industrial Microbiology & Biotechnology·Yihan LiuFuping Lu
Dec 17, 2014·Trends in Pharmacological Sciences·Jeffrey W KeillorAbdullah Akbar
May 3, 2015·Molecular Cancer Research : MCR·Matthew L FisherCandace Kerr
Sep 16, 2015·BMC Biotechnology·Barbara SalisRodolfo Schrepfer
Jan 21, 2016·Journal of Biomedical Science·Chien-Hsun WuHan-Chung Wu
Jul 29, 2017·The Journal of Biological Chemistry·Wojtek SteffenMichael Schraeml

❮ Previous
Next ❯

Citations

Sep 28, 2018·Chemical Society Reviews·Yi ZhangMark D Distefano
Jan 4, 2020·World Journal of Microbiology & Biotechnology·Lovaine DuarteMarco Antônio Záchia Ayub
Mar 17, 2020·World Journal of Microbiology & Biotechnology·N DotiM Ruvo
Feb 5, 2019·Micromachines·Maria Pia SavocaElisabetta A M Verderio

❮ Previous
Next ❯

Methods Mentioned

BETA
deamidation
FRET
fluorescence spectroscopy
peptide library
flow cytometry
FACS
protein assay
Fluorescence

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.