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 alpha-factor precursor was expressed in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature alpha-factor peptide. This enabled the use of a simple bioassay (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 endoprotease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues. Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin. The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography. Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin.
Bourbonnais
Y
Y
Département de biochimie et de microbiologie, la Structure et l'Ingénierie des Protéines (CREFSIP), niversité Laval, Quebec City, Canada G1K 7P4.
Larouche
C
C
Tremblay
G M
GM
D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011498:Protein Precursors / Q000235:genetics; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D015842:Serine Proteinase Inhibitors
D011498:Protein Precursors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015842:Serine Proteinase Inhibitors; D010196:Pancreatic Elastase; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae