Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies.
We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.
Richter
Wito
W
Institute of Biochemistry, Medical Faculty, Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, Leipzig, Germany.
Hermsdorf
Thomas
T
Kronbach
Thomas
T
Dettmer
Dietrich
D
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification*; D017398:Alternative Splicing; D015153:Blotting, Western; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D054708:Cyclic Nucleotide Phosphodiesterases, Type 7; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism*; D007477:Ions; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008957:Models, Genetic; D011485:Protein Binding; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D019163:Reducing Agents / Q000494:pharmacology; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D015854:Up-Regulation
D018076:DNA, Complementary; D007477:Ions; D007527:Isoenzymes; D011994:Recombinant Proteins; D019163:Reducing Agents; D006493:Heparin; D012685:Sepharose; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054708:Cyclic Nucleotide Phosphodiesterases, Type 7; C517458:PDE7A protein, human