Heterologous expression and purification of recombinant rolipram-sensitive cyclic AMP-specific phosphodiesterases.
With the cloning of cDNAs coding for the different phosphodiesterase 4 (PDE4) isoenzymes present in mammals, homogeneous preparations of these forms have become readily available. This strategy has greatly facilitated the understanding of the properties of the myriad of isoforms derived from the four PDE4 genes found in mammals, and has opened a new avenue to develop inhibitors with a different degree of selectivity for each isoform. Here we describe the strategies and methods used to express PDE4 in bacterial, yeast, insect, and mammalian cell heterologous systems, and review the advantages and disadvantages of each of these expression strategies. In addition, procedures to purify the recombinant proteins are described. The recently developed purification of a PDE4 by immunoaffinity chromatography provides a rapid and efficient method to prepare large quantities of PDE4. This method should be very useful for structural and kinetic studies on the PDE4D isoforms.
Salanova
M
M
Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317, USA.
Jin
S C
SC
Conti
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification*; D020889:Rolipram; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018411:Spodoptera / Q000235:genetics
D011760:Pyrrolidinones; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D020889:Rolipram