A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A₂ as a model

An active preparation of human phospholipase A₂ (PLA₂) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA₂ isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA₂ from the fusion protein. The fusion protein was composed of a β-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA₂. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA₂ was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A₂.