Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase

The Fc‐III tag is a newly developed fusion tag that can be applied to protein purification and detection. In the present work, we use the Fc‐III‐tagged green fluorescent protein (GFP) and human muscle creatine kinase (CK) as model systems to investigate effects of the Fc‐III tag on activities and stabilities of the expressed multicysteine‐containing proteins. Our results show 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. The activity and stability of the Fc‐III‐tagged CK is slightly lower than that of the tag‐free CK, but is higher than that of the His‐tagged CK 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 was proposed, which may provide insights into the effect of the Fc‐III tag on the conformations of disulfide‐bridged proteins.