Expression of a synthetic gene encoding potato carboxypeptidase inhibitor using a bacterial secretion vector.
A synthetic gene encoding the 39-amino-acid (aa) potato carboxypeptidase inhibitor IIa (PCI-IIa) has been constructed and expressed using the secretion vector, pIN-III-ompA-3, fused in frame to the OmpA signal peptide-encoding sequence. Recombinant Escherichia coli secreted a PCI with 10 additional aa at the N terminus (rePCI + 10). These extra aa were removed by site-directed mutagenesis giving a PCI with no additional aa (rePCI), as shown by fast atom bombardment mass spectrometry (M(r) 4295). The two forms of rePCI were found almost exclusively in the culture medium, not in the periplasmic space, as would be expected from OmpA signal peptide fusions. Both rePCI + 10 and rePCI are biologically active and react strongly with serum raised against PCI from potato. A method for the purification of rePCI to homogeneity has been developed. The purified rePCI shows a Ki for carboxypeptidase A within the range of the natural PCI-IIa (1.5-2.7 nM). These results indicate that both rePCI + 10 and rePCI are properly folded and that their three disulfide bridges are correctly formed. Together with previous reports, our results show that fusion to a secretion signal peptide is an effective way of producing small proteins containing disulfide bridges in a biologically active form.
Molina
M A
MA
Institut de Biologia Fonamental, Universitat Autónoma de Barcelona, Spain.
Avilés
F X
FX
Querol
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005813:Genes, Synthetic / Q000235:genetics*; D005822:Genetic Vectors / Q000502:physiology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011198:Solanum tuberosum / Q000235:genetics*
D010940:Plant Proteins; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002268:Carboxypeptidases