title: Use of Vibrio spp. for expression of Escherichia coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes simplex virus type 1.
abstract: The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198-204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.
authors.affiliation: Institute of Microbiology, University of Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Loregian
authors.forename: T R
authors.initials: TR
authors.lastname: Hirst
authors.forename: H S
authors.initials: HS
authors.lastname: Marsden
authors.forename: G
authors.initials: G
authors.lastname: Palù
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics*; D003470:Culture Media; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D004492:Edetic Acid; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics*; D005677:G(M1) Ganglioside / Q000737:chemistry; D015684:Gene Products, pol / Q000737:chemistry; D018259:Herpesvirus 1, Human / Q000201:enzymology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D015684:Gene Products, pol; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005677:G(M1) Ganglioside; D004492:Edetic Acid; C027538:heat-labile enterotoxin, E coli; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus