Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional Yapsin 1 (Yps1p) endoprotease

Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family. A chimeric gene encoding polyhistidine -tagged human pre-elafin fused to the yeast a-factor precursor was expresse d in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature a-factor peptide. This enabled the use of a simple bioas say (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule, We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endopr otease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subse t of single lysine residues. Expression in a yapsin l-deficient yeast strai n was an indispensable condition to allow the efficient production of full- length human pre-elafin. The recombinant inhibitor was purified from concen trated culture medium by ammonium sulfate precipitation, affinity purificat ion on a Ni2+ resin, and cation exchange chromatography, Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward d ifferent serine proteases to that reported for mature elafin. (C) 2000 Acad emic Press.