The cysteine residues of recombinant human gastric lipase

Recombinant human gastric lipase (rHGL) and three of its cysteine mutants ( cysteine 227, 236, and 244 substitued for threonine or serine) were express ed in the baculovirus/insect cell system and purified to homogeneity by per forming a two-step procedure, Substituting Ser for Cys 227 and Cys 236 resu lted in mutant lipases with a significantly lower level of activity (30% an d 22%, respectively) on a short chain triglyceride (tribuyrin) substrate, w hile the mutation at position 244 only slightly reduced the activity. Using 4, 4'-dithiopyridine (4-PDS) as a sulfhydryl reagent on the above mutants, it was possible to clearly identify the single sulfhydryl residue at posit ion 244 and consequently, the disulfide bridge at position 227-236, No pote ntial disulfide bridges were formed during the protein folding between cyst eines 227-244 or between cysteines 236-244, as thought to occur in the case of rabbit gastric lipase (RGL). The present results are consistent with th e recently determined 3D-structure of rHGL. (C) 1999 Academic Press.