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Literature summary for 2.7.3.2 extracted from

  • Feng, S.; Gong, Y.; Adilijiang, G.; Deng, H.
    Effects of the Fc-III tag on activity and stability of green fluorescent protein and human muscle creatine kinase (2013), Protein Sci., 22, 1008-1015.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of Fc-III-tagged enzyme and of His-tagged enzyme in Escherichia coli strain BL21 (DE3)-plysS, respectively, the Fc-III-tagged GFP-fusion muscle creatine kinase is used as a model system to investigate effects of the Fc-III tag on activities and stabilities of recombinantly expressed multicysteine-containing proteins Homo sapiens

Protein Variants

Protein Variants Comment Organism
additional information construction of a Fc-III-tagged GFP fusion muscle creatine kinase (CK) as a model system to investigate effects of the Fc-III tag on activities and stabilities of recombinantly expressed multicysteine-containing proteins. The Fc-III tag has no adverse effects on the fluorescence of GFP and reduces the occurrence of GFP misfolding due to incorrect Cys oxidation compared with the His-tagged protein. Activity and stability of the Fc-III-tagged creatine kinase is slightly lower than that of the tag-free creatine kinase, but is higher than that of the His-tagged creatine kinase as determined by the ratio of the oxidized versus reduced CK. A major portion of His-tagged CK is in its oxidized form, while that of the Fc-III-tagged CK is in its reduced form. A folding model of CK with different tags is proposed, overview. The decrease of the activity of CK may attribute to the tag-induced changes of the tertiary structures of the targeted proteins Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
guanidinium hydrochloride 0.1-3.0 M, under both conditions, the tag-free enzyme shows the lowest degree of aggregation, followed by His-tagged CK, and Fc-III-tagged CK has the highest degree of aggregation Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + creatine Homo sapiens
-
ADP + phosphocreatine
-
r

Organism

Organism UniProt Comment Textmining
Homo sapiens P06732
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant Fc-III-tagged enzyme from Escherichia coli strain BL21 (DE3)-plysS by IgG-Fc immunoaffinity chromatography, recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3)-plysS by nickel affinity chromatography Homo sapiens

Source Tissue

Source Tissue Comment Organism Textmining
skeletal muscle
-
Homo sapiens
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + creatine
-
Homo sapiens ADP + phosphocreatine
-
r

Subunits

Subunits Comment Organism
More formation of the disulfide bond between the C74 and the C146 residues in the oxidized form Homo sapiens

Temperature Optimum [°C]

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

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
52
-
the recombinant enzymes are stable upto 50°C, the recombinant His-tagged and Fc-III-tagged enzymes show similar heat-resistance and are less stable than that of the tag-free enzyme. They start to lose their activities at 52°C and are relatively less active at the same denaturing temperature than tag-free enzyme. The DELTAT0.5 value of the tag-free enzyme is about 58°C and that of the tagged enzyme is around 57°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
ATP
-
Homo sapiens

General Information

General Information Comment Organism
additional information formation of the disulfide bond between the C74 and the C146 residues in the oxidized form Homo sapiens