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

Abstract An active preparation of human phospholipase A 2 (PLA 2 ) was made after expression as an insoluble fusion protein in Escherichia coli . The new key elements required for PLA 2 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 PLA 2 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 PLA 2 . 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 PLA 2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A 2 .