Eugen Schnabel

Semisynthetic engineering of proteinase inhibitor homologues.

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.

Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli

As a basis for a protein design project, we decided to produce the human pancreatic secretory trypsin inhibitor (PSTI) in its active form. Total gene synthesis was carried out efficiently by (i) computer design of the gene fragments, (ii) synthesis of the oligodeoxynucleotides by the segmental support method, and (iii) assembly of double strands under optimized ligation conditions. Fusion to the ompA gene signal peptide led to secretion of processed PSTI in various constructions, with or without additional amino acids (aa) at the N-terminus. The secreted proteins (56 to 63 aa) were biologically active, suggesting that the three cysteine bridges were correctly formed. Surprisingly, after induction the product was found almost exclusively in the culture medium. Variants of PSTI with Asp or Asn at aa positions 21 and 29 [sequences published by Greene et al., Methods Enzymol. (1976) 813-825, and by Yamamoto et al., Biochem. Biophys. Res. Commun. (1985) 605-612] showed the same Ki for both human and porcine trypsin.

Eine Synthese der B-Kette des Insulins

Die B-Kette des Insulins wurde in Form des Derivates Carbobenzoxy-L-phenylalanyl-L-valyl-L-asparaginyl-L-glutaminyl-Nim-benzyl-L-histidyl-L-leucyl-S-benzyl-L-cysteinyl-glycyl-L-seryl-L-histidyl- L-leucyl- L–valyl-L-glutamyl-L-alanyl-L-leucyl- L-tyrosyl-L-leucyl- L-valyl - S - benzyl-L-cysteinyl-glycyl - L- glutamyl - NG-tosyl-L-arginyl-glycyl-L-phenylalanyl-L-phenylalanyl-L-tyrosyl-L-threonyl-L-prolyl- Nε · tosyl- L- lysyl - L- alanin aus den Teilstücken B 1—8, B 9 — 14, B 15 — 20 und B 21 — 30 synthetisiert. Die Schutzgruppenfreie Polypeptidkette lieferte nach der Verknüpfung mit synthetischer und natürlicher Schafinsulin-A-Kette Insulin-aktive Präparate.

Neusynthese der Insulinsequenz B 21-30

Die Insulinsequenz B 21 - 30 wurde in Form des Derivates Z-Glu (OBut) -Arg (Tos) -Gly-Phe-Phe-Tyr-Thr-Pro-Lys (Tos) -Ala-OBut synthetisiert.