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 | 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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + creatine | Homo sapiens | - |
ADP + phosphocreatine | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P06732 | - |
- |
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 | Comment | Organism | Textmining |
---|---|---|---|
skeletal muscle | - |
Homo sapiens | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + creatine | - |
Homo sapiens | ADP + phosphocreatine | - |
r |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Homo sapiens |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Homo sapiens |
General Information | Comment | Organism |
---|---|---|
additional information | formation of the disulfide bond between the C74 and the C146 residues in the oxidized form | Homo sapiens |