Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core.
We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B). High level expression in Escherichia coli has been achieved with yields of up to 20mg/L. The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand. PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM. Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B. The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674. Constructs starting at E665 and M674 were fully active and devoid of activity, respectively. A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected. This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667. Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.
Varnerin
Jeffrey P
JP
Department of Metabolic Disorders, Merck and Co., Rahway, NJ 07065, USA.
Chung
Christine C
CC
Patel
Sangita B
SB
Scapin
Giovanna
G
Parmee
Emma R
ER
Morin
Nancy R
NR
MacNeil
Douglas J
DJ
Cully
Doris F
DF
Van der Ploeg
Lex H T
LH
Tota
Michael R
MR
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517291:PDE3B protein, human