Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 308, Issue 1, Pages 87-93Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0006-291X(03)01340-8
Keywords
calcineurin; fusion; phosphatase activity; folding; binding site
Categories
Ask authors/readers for more resources
Calcineurin (CN) is a Ca2+/calmodulin(CaM)-dependent serine/threonine protein phosphatase which is a heterodimer composed of a 61 kDa catalytic subunit (CNA) and a 19 kDa regulatory subunit (CNB). The enzyme is critical for several important intracellular signal-transducing pathways, including T-cell activation. Its crystal structure reveals that the C-terminal of CNB lies in close vicinity of the N-terminal of CNA and each end has a long arm not involved in the active site. After fusing two subunits, it was determined that folding and function of the protein were not affected by the fusion. We amplified a fused gene of A and B subunits using a pair of linker primers including six codons of glycine. A single chain calcineurin was constructed and purified to near-homogeneity. The recombinant enzyme was fully soluble, displayed high specific activity with substrate, and exhibited biochemical properties and kinetic parameters similar to those of the native enzyme from the bovine brain. It was still activated by Ca2+/calmodulin but was not regulated by extra CNB and was still strongly stimulated by Mn2+ and Ni2+ divalent metal ions. The solution conformations of both recombinant enzyme and bovine calcineurin were assayed under the same conditions using intrinsic fluorescence spectroscopy and circular dichroism spectropolarimetry, and results showed their graphs are approximately identical. Our findings suggested that the fusion of A and B subunits of calcineurin does not affect their folding pathways and structural changes involved in their function, furthermore, they are bound to the correct binding site. (C) 2003 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available