A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm.
Human pepsinogen is the secreted inactive precursor of pepsin. Under the acidic conditions present in the stomach it is autocatalytically cleaved into the active protease. Pepsinogen contains three consecutive disulfides, and was used here as a model protein to investigate the production of aspartic proteases in the Escherichia coli periplasm. Various N-terminal translocation signals were applied and several different expression vectors were tested. After fusion to pelB, dsbA or ompT signal peptides no recombinant product could be obtained in the periplasm using the T7 promoter. As a new approach, human pepsinogen was fused to E. coli ecotin (E. coli trypsin inhibitor), which is a periplasmic homodimeric protein of 142 amino acids per monomer containing one disulfide bridge. The fusion protein was expressed in pTrc99a. After induction, the ecotin-pepsinogen fusion protein was translocated into the periplasm and the ecotin signal peptide was cleaved. Upon acid treatment, the fusion protein was converted into pepsin, indicating that pepsinogen was produced in its native form. In shake flasks experiments, the amount of active fusion protein present in the periplasm was 100 microg per litre OD 1, corresponding to 70 microg pepsinogen. After large scale cultivation, the fusion protein was isolated from the periplasmic extract. It was purified to homogeneity with a yield of 20%. The purified protein was native. Acid-induced activation of the fusion protein proceeded very fast. As soon as pepsin was present, the ecotin part of the fusion protein was rapidly digested, followed by a further activation of pepsinogen.
Malik
Ajamaluddin
A
Institut für Biotechnologie, Martin-Luther Universität Halle, Germany.
Rudolph
Rainer
R
Söhling
Brigitte
B
D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D020116:Pepsinogen A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019897:Periplasm / Q000235:genetics; D033903:Periplasmic Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D020116:Pepsinogen A