Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein.
A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004247:DNA / Q000235:genetics*; D010450:Endopeptidases / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D010435:Pepsinogens / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D013552:Swine; D014170:Transformation, Genetic
D003470:Culture Media; D010435:Pepsinogens; D021382:Protein Sorting Signals; D004247:DNA; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases