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 beta-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.
Tseng
A
A
Pacific Biotechnology, Ltd., Ruschutters Bay, NSW, Australia.
Buchta
R
R
Goodman
A E
AE
Loughnan
M
M
Cairns
D
D
Seilhamer
J
J
Johnson
L
L
Inglis
A S
AS
Scott
K F
KF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D054467:Phospholipases A2; D010957:Plasmids; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D000596:Amino Acids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004247:DNA; D010741:Phospholipases A; D054467:Phospholipases A2