Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli.
A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.
Institute of Biochemistry, Medical Faculty, University of Leipzig, Liebigstrasse 16, Leipzig, D-04103, Germany.
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W
Richter
T
T
Hermsdorf
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H
Lilie
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U
Egerland
R
R
Rudolph
T
T
Kronbach
D
D
Dettmer
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human