Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins.
Aspartic Acid
/ chemistry
Biocatalysis
Biochemistry
/ methods
Carbon Isotopes
Cysteine
/ chemistry
Electrophoresis, Polyacrylamide Gel
/ methods
Escherichia coli
/ chemistry
Exteins
Gene Expression
Genetic Vectors
/ genetics
Histidine
/ chemistry
Hydrogen-Ion Concentration
Inteins
Maltose-Binding Proteins
/ chemistry
Nitrogen Isotopes
Nuclear Magnetic Resonance, Biomolecular
/ methods
Protein Splicing
Recombinant Fusion Proteins
/ biosynthesis
Intein
NMR spectroscopy
Protein overexpression
Protein splicing
SDS-PAGE
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
8
3
2020
pubmed:
8
3
2020
medline:
6
3
2021
Statut:
ppublish
Résumé
The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.
Identifiants
pubmed: 32144663
doi: 10.1007/978-1-0716-0434-2_4
pmc: PMC7325523
mid: NIHMS1594922
doi:
Substances chimiques
Carbon Isotopes
0
Maltose-Binding Proteins
0
Nitrogen Isotopes
0
Recombinant Fusion Proteins
0
Aspartic Acid
30KYC7MIAI
Histidine
4QD397987E
Cysteine
K848JZ4886
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
55-73Subventions
Organisme : NCI NIH HHS
ID : R01 CA206592
Pays : United States
Organisme : NIGMS NIH HHS
ID : R15 GM132817
Pays : United States
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