Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein. | - CCMAR -

Journal Article

TítuloProduction and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein.
Publication TypeJournal Article
AuthorsAnjos, L, Rotllant, J, Guerreiro, PM, Hang, X, Canario, AVM, Balment, R, Power, DM
Year of Publication2005
JournalGen Comp Endocrinol
Date Published2005 Aug
Palavras-chaveAmino Acid Sequence, Animals, Blotting, Western, Cyclic AMP, DNA, Complementary, Electrophoresis, Polyacrylamide Gel, Genetic Vectors, Molecular Sequence Data, Parathyroid Hormone-Related Protein, Recombinant Proteins, Sea Bream, Sequence Alignment

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1-125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1-125) synthesis was induced by addition of 1mM isopropyl-beta-d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1-125) as judged by SDS-PAGE and yielded up to 40mg/L of culture medium (3.3mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1-125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1-34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled (125)I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.


Alternate JournalGen. Comp. Endocrinol.
PubMed ID15993105
CCMAR Authors