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 (Nakakubo et al., 2000), whereby recombinant h-chymase (rh-chymase) was secreted as a mature form with the correct N-terminal amino acid sequence and was easily purified. In the course of investigation of secretory rh-chymase, we also found large amounts of chymase to be present in insoluble form in the transformant cell. Although the cellular rh-chymase had no proteolytic activity, its chymotryptic activity was restored in a reconstitution process utilizing guanidine and glutathione. As with secretory rh-chymase, efficient purification was possible by heparin affinity chromatography. The purified cellular rh-chymase showed the same mobility as secretory rh-chymase in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before and after deglycosylation. N-terminal amino acid sequence analysis revealed that the signal peptide had been correctly removed. K(m) value (5.93 mM), as well as pH profile and inhibition profile toward protease inhibitors of reconstituted cellular rh-chymase, indicated that the rh-chymase enzymatically closely resembles native h-chymase. Furthermore, it showed a greatly restricted proteolytic activity towards Ang I, and formed Ang II without the further cleavage which is a feature of h-chymase. It was thus found that the insoluble rh-chymase stored in the cells could be solubilized and reconstituted to give the same structure as h-chymase, not only in terms of enzyme active site but also of substrate recognition site.
Nakakubo
H
H
Drug Discovery Laboratories, Welfide Corporation, 2-25-1, Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan.
Morita
M
M
Imada
T
T
Takai
S
S
Shiota
N
N
Miyazaki
M
M
Nakamura
N
N
D000581:Amidohydrolases / Q000737:chemistry; D000803:Angiotensin I / Q000737:chemistry; D015153:Blotting, Western; D002847:Chromatography, Agarose; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000201:enzymology*; D011480:Protease Inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics
D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D000803:Angiotensin I; D006596:Hexosaminidases; D012697:Serine Endopeptidases; D053818:Chymases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase