Site-specific alteration of cysteine 176 and cysteine 234 in the lactose carrier of Escherichia coli.

AbstractSynaptic transmission leading to release of neurotransmitters in the nervous system is a fast and highly dynamic process. Previously, protein interaction and phosphorylation have been thought to be the main regulators of synaptic transmission. Here we show a novel potential modulator of synaptic transmission, sialylation of N-linked glycosylation. The negatively charged sialic acids can be modulated, similarly to phosphorylation, by the action of sialyltransferases and sialidases thereby changing local structure and function of membrane glycoproteins. We characterized site-specific alteration in sialylation on N-linked glycoproteins in isolated rat nerve terminals after brief depolarization using quantitative sialiomics. We identified 1965 formerly sialylated N-linked glycosites in synaptic proteins and found that the abundances of 430 glycosites changed after five seconds depolarization. We observed changes on essential synaptic proteins such as synaptic vesicle proteins, ion channels and transporters, neurotransmitter receptors and cell adhesion molecules. This study is to our knowledge the first to describe ultra-fast site-specific modulation of the sialiome after brief stimulation of a biological system.

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Site-specific DNA cleavage of synthetic NarL sites by an engineered Escherichia coli NarL protein-1,10-phenanthroline cleaving agent

Protein Science ◽

10.1110/ps.0212502 ◽

2008 ◽

Vol 11 (10) ◽

pp. 2427-2436 ◽

Cited By ~ 9

Author(s):

Gaoping Xiao ◽

Daniel L. Cole ◽

Robert P. Gunsalus ◽

David S. Sigman ◽

Chi-Hong B. Chen

Keyword(s):

Escherichia Coli ◽

Dna Cleavage ◽

Site Specific ◽

Engineered Escherichia Coli

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The Site-Specific Recombination System of the Escherichia coli Bacteriophage Φ24B

Frontiers in Microbiology ◽

10.3389/fmicb.2020.578056 ◽

2020 ◽

Vol 11 ◽

Author(s):

Mohammed Radhi Mohaisen ◽

Alan John McCarthy ◽

Evelien M. Adriaenssens ◽

Heather Elizabeth Allison

Keyword(s):

Escherichia Coli ◽

Site Specific ◽

Site Specific Recombination ◽

Recombination System ◽

Site Specific Recombination System

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Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽

10.3390/biom9070255 ◽

2019 ◽

Vol 9 (7) ◽

pp. 255 ◽

Cited By ~ 16

Author(s):

Sviatlana Smolskaya ◽

Yaroslav Andreev

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Nonsense Suppression ◽

Expression Vectors ◽

Site Specific ◽

Suppressor Trna ◽

E Coli ◽

Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

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