Functional reconstitution of an active recombinant human chymase from Pichia pastoris cell lysate

We have previously reported efficient production of mature human chymase (h -chymase) using an original system of expression in Pichia pastoris (Nakaku bo et al,, 2000), whereby recombinant h-chymase (rh-chymase) was secreted a s a mature form with the correct N-terminal amino acid sequence and was eas ily purified. In the course of investigation of secretory rh-chymase, me al so found large amounts of chymase to be present in insoluble form in the tr ansformant cell. Although the cellular rh-chymase had no proteolytic activi ty, its chymotryptic activity was restored in a reconstitution process util izing guanidine and glutathione, As with secretory rh-chymase, efficient pu rification mas possible by heparin affinity chromatography. The purified ce llular rh-chymase showed the same mobility as secretory rh-chymase in sodiu m dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after deglycosylation. N-terminal amino acid sequence analysis revealed th at the signal peptide had been correctly removed, K-m value (5.93 mM), as w ell as pH profile and inhibition profile toward protease inhibitors of reco nstituted cellular rh-chymase, indicated that the rh-chymase enzymatically closely resembles native h-chymase. Furthermore, it showed a greatly restri cted proteolytic activity towards Ang I, and formed Ang II without the furt her cleavage which is a feature of h-chymase. It was thus found that the in soluble rh-chymase stored in the cells could be solubilized and reconstitut ed to give the same structure as h-chymase, not only in terms of enzyme act ive site but also of substrate recognition site. Copyright (C) 2000 John Wi ley & Sons, Ltd.