Heterologous expression of a gene that codes for Pg8, a scorpion toxin of Parabuthus granulatus, capable of generating protecting antibodies in mice.
A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.
García-Gómez
Blanca I
BI
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Olamendi-Portugal
Timoteo C
TC
Paniagua
Jorge
J
van der Walt
Jurg
J
Dyason
Karin
K
Possani
Lourival D
LD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis; D000997:Antivenins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005796:Genes / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000201:enzymology / Q000235:genetics* / Q000276:immunology*; D012605:Scorpions / Q000235:genetics*
D000906:Antibodies; D000997:Antivenins; D018076:DNA, Complementary; C543526:Pg8 protein, Parabuthus granulatus; D011994:Recombinant Proteins; D012604:Scorpion Venoms