Reconstitution of active human calcineurin from recombinant subunits expressed in bacteria.
Calcineurin, a protein phosphatase found in eukaryotic cells, presents a challenging problem in heterologous protein expression because it is both heterodimeric and posttranslationally modified. In this paper, we describe the cloning of both subunits (catalytic A and regulatory B) of calcineurin from a human cDNA library and their expression at high levels in Escherichia coli. The calcineurin A subunit is expressed as an insoluble glutathione S-transferase fusion protein, while the calcineurin B subunit is soluble upon direct expression. Catalytically active holoenzyme is derived from the separately expressed subunits using a three-step refolding protocol. First, the fusion protein is solubilized, then it is cleaved at the fusion junction with thrombin, and, finally, a catalytically competent calcineurin A:calcineurin B:calmodulin complex is reconstituted by cofolding the separately purified components. In addition, we show that a similar refolding protocol can be applied to a C-terminally truncated form of calcineurin A, which lacks an autoinhibitory and calmodulin-binding domain.
Department of Biophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-9500, USA.
L L
LL
Rokosz
S J
SJ
O'Keefe
J N
JN
Parsons
P M
PM
Cameron
J J
JJ
Burbaum
D000595:Amino Acid Sequence; D001483:Base Sequence; D019703:Calcineurin; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D010865:Pilot Projects; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D013917:Thrombin