Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Side reaction in peptide synthesis. Formation of oxazolidone derivatives

2-Oxazolidone derivatives formed through an intramolecular reaction in the process of alkaline treatment of urethane-type N-protected peptides of which the N-terminal residues were Ser or Thr having unprotected hydroxyl groups. In oder to avoid this side reaction, the esters of these peptides could be cleaved by enzymatic hydrolyses instead of saponification.