Recombinant eggshell ovocalyxin-32: expression, purification and biological activity of the glutathione S-transferase fusion protein.
The avian eggshell is a highly ordered biomineral composed mainly of calcium carbonate associated with an organic matrix composed of proteins, glycoproteins and proteoglycans. This structure provides the developing embryo with protection from physical damage and microbial invasion. Ovocalyxin-32 (OCX-32) is a 32 kDa eggshell-specific matrix protein which has been cloned and demonstrates 30% identity with the mammalian carboxypeptidase inhibitor, latexin. In order to further study its function, recombinant OCX-32 protein was expressed in E. coli. The protein was extracted from inclusion bodies and purified by sequential DEAE Sepharose and Ni2+ metal ion affinity chromatographies as a 58 kDa GST-fusion protein. The refolded GST-OCX-32 significantly inhibited bovine carboxypeptidase and also inhibited the growth of Bacillus subtilis. The results suggest that OCX-32 may show similar activity to the fusion protein and reinforce the antimicrobial properties of the eggshell by providing protection to the developing avian embryo. OCX-32 is the first example of an eggshell specific protein to be successfully cloned and expressed in a prokaryotic system. The association of an antimicrobial protease inhibitor with the outer eggshell and cuticle of the table egg may enhance the food safety of this product.
Xing
Jun
J
Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5.
Wellman-Labadie
Olivier
O
Gautron
Joel
J
Hincke
Maxwell T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers; D004527:Egg Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004527:Egg Proteins; D011993:Recombinant Fusion Proteins; C439069:ovocalyxin 32; D005982:Glutathione Transferase