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.