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.
Maywald
F
F
Department of Genetics, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, F.R.G.
Böldicke
T
T
Gross
G
G
Frank
R
R
Blöcker
H
H
Meyerhans
A
A
Schwellnus
K
K
Ebbers
J
J
Bruns
W
W
Reinhardt
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005796:Genes; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000235:genetics*
D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014357:Trypsin