Literature summary for 3.6.4.B7 extracted from

Liu, J.; Ekanayake, O.; Santoleri, D.; Walker, K.; Rozovsky, S.
Efficient generation of hydrazides in proteins by RadA split intein (2019), ChemBioChem, 20, 1-8 .

Search for more literature for 3.6.4.B7

Application

Application	Comment	Organism
synthesis	efficient generation of hydrazides in proteins by RadA split intein	Pyrococcus horikoshii

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of codon-optimized MBP-tagged and Strep-II-tagged wild-type and mutant RadAs proteins in Escherichia coli strain BL21(DE3)	Pyrococcus horikoshii

Protein Variants

Protein Variants	Comment	Organism
additional information	construction of enzyme mutants MBP-RadAC46, MBP-Arg-RadA, MBP-Glu-RadA, MBP-Asn-RadA, MBP-Pro-RadA, MBP-Asp-RadA, MBP-Ser-RadA, MBP-Cys-RadA, MBP-Gly-RadA, MBP-Arg-RadADELTAC46, MBP-Glu-RadADELTAC46, MBP-Asn-RadADELTAC46, MBP-Pro-RadADELTAC46, MBP-Asp-RadADELTAC46, MBP-Ser-RadADELTAC46, MBP-Cys-RadADELTAC46, and MBP-Gly-RadADELTAC46	Pyrococcus horikoshii

Organism

Organism	UniProt	Comment	Textmining
Pyrococcus horikoshii	O58001	-	-
Pyrococcus horikoshii ATCC 700860	O58001	-	-
Pyrococcus horikoshii DSM 12428	O58001	-	-
Pyrococcus horikoshii JCM 9974	O58001	-	-
Pyrococcus horikoshii NBRC 100139	O58001	-	-
Pyrococcus horikoshii OT-3	O58001	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
proteolytic modification	the protein undergoes a protein self splicing that involves a post-translational excision of the intervening region (intein) followed by peptide ligation	Pyrococcus horikoshii

Purification (Commentary)

Purification (Comment)	Organism
recombinant MBP-tagged RadA from Escherichia coli strain BL21(DE3) by amylose affinity chromatography, removal of the MBP-tag, all constructs contain a tobacco etch virus (TEV) protease cleavage site (ENLYFQS) following the MBP	Pyrococcus horikoshii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii	?	-	-
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii DSM 12428	?	-	-
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii NBRC 100139	?	-	-
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii JCM 9974	?	-	-
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii ATCC 700860	?	-	-
additional information	to generate C-terminal hydrazides in proteins, an efficient intein-based preparation method is developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method is expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. The versatile preparation method can expand the utilization of protein C-terminal hydrazides in protein preparation and modification. Method evaluation, overview. RadA, which had appreciable splicing efficiency with non-native extein residues. RadA intein is more tolerant to the nature of the residue at the -1 position, its rate of cleavage is slower than that of the VMA and GyrA inteins. Hence, its splicing typically requires conditions such as high temperature or partial denaturation. Generalizability of the method	Pyrococcus horikoshii OT-3	?	-	-

Synonyms

Synonyms	Comment	Organism
DNA repair and recombination protein	UniProt	Pyrococcus horikoshii
RadA	-	Pyrococcus horikoshii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Pyrococcus horikoshii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Pyrococcus horikoshii

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Release 2023.1 (January 2023)