Journal Article
Título | Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein. |
Publication Type | Journal Article |
Authors | Anjos, L, Rotllant, J, Guerreiro, PM, Hang, X, Canario, AVM, Balment, R, Power, DM |
Year of Publication | 2005 |
Journal | Gen Comp Endocrinol |
Volume | 143 |
Questão | 1 |
Date Published | 2005 Aug |
Pagination | 57-65 |
ISSN | 0016-6480 |
Palavras-chave | Amino 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 |
Abstract | 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. |
DOI | 10.1016/j.ygcen.2005.02.020 |
Sapientia | |
Alternate Journal | Gen. Comp. Endocrinol. |
PubMed ID | 15993105 |