Literature summary for 2.7.7.B22 extracted from

Voigt, F.; Wiedemann, L.; Zuliani, C.; Querques, I.; Sebe, A.; Mates, L.; Izsvak, Z.; Ivics, Z.; Barabas, O.
Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering (2016), Nat. Commun., 7, 11126.

Search for more literature for 2.7.7.B22

Application

Application	Comment	Organism
molecular biology	Sleeping Beauty is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally thioredoxinA-6xHis-tagged wild-type and mutant enzymes in Escherichia coli strain RosettaII(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
puriifed recombinant tagged transposase catalytic domain SB100X, residues 114-340, by hanging drop vapour diffusion method, mixing of 0.002 ml of 19 mg/ml protein in 20 mM HEPES, pH 7.2, 50 mM NaCl, 2% v/v glycerol, 10 mM MgCl2, and 0.2 mM TCEP, with 0.002 ml of precipitant solution containing 0.1 M Na-citrate, pH 4.8, and 3.2 M ammonium sulfate, 4°C, 5 days, X-ray diffraction structure determination and analysis at 1.40 A resolution, PDB ID 3HOS	Homo sapiens

Crystallization (Comment)

Organism

puriifed recombinant tagged transposase catalytic domain SB100X, residues 114-340, by hanging drop vapour diffusion method, mixing of 0.002 ml of 19 mg/ml protein in 20 mM HEPES, pH 7.2, 50 mM NaCl, 2% v/v glycerol, 10 mM MgCl2, and 0.2 mM TCEP, with 0.002 ml of precipitant solution containing 0.1 M Na-citrate, pH 4.8, and 3.2 M ammonium sulfate, 4°C, 5 days, X-ray diffraction structure determination and analysis at 1.40 A resolution, PDB ID 3HOS

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
E279D	a catalytically inactive transposase mutant	Homo sapiens
K339	site-directed mutagenesis, the mutant shows 20% reduced activity compared to the wild-type enzyme	Homo sapiens
additional information	design of two additional hyperactive transposase variants using the enzyme crystal structure, evaluation of structure-based engineering of tailored SB transposases, overview	Homo sapiens
N280	site-directed mutagenesis, the mutant shows 50% reduced activity compared to the wild-type enzyme	Homo sapiens
N280/K339	site-directed mutagenesis, almost catalytically inactive mutant	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally thioredoxinA-6xHis-tagged wild-type and mutant enzymes from Escherichia coli strain RosettaII(DE3) by nickel affinity chromatography	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	Sleeping Beauty transposase excision activity is quantified using a fluorescence-activated cell sorting (FACS)-based excision assay in HeLa cells, overall SB transposition activity is also detected in HeLa cells	Homo sapiens	?	-	?

Subunits

Subunits	Comment	Organism
More	the SB100X catalytic domain assumes an RNaseH-fold with all catalytic residues assembled in the active site, it contains conserved alpha-helices and beta-strands, structure modeling of the SB transposase/transposon end/target DNA complex using crystal structure, PDB ID 3HOS, overview	Homo sapiens

Synonyms

Synonyms	Comment	Organism
sleeping beauty transposase	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.2	-	assay at	Homo sapiens

General Information

General Information	Comment	Organism
evolution	Sleeping Beauty is a prominent Tc1/mariner superfamily DNA transposon, mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends	Homo sapiens
additional information	modeling of the SB transposase/transposon end/target DNA complex, overview	Homo sapiens