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

• Published in: General and comparative endocrinology 2005-08, Vol.143 (1), p.57-65
• Main Authors: Anjos, L., Rotllant, J., Guerreiro, P.M., Hang, X., Canario, A.V.M., Balment, R., Power, D.M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biological activity; Blotting, Western; Cyclic AMP - metabolism; DNA, Complementary - metabolism; E. coli production; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Vectors; Immunological activity; Marine; Molecular Sequence Data; Parathyroid Hormone-Related Protein - biosynthesis; Parathyroid Hormone-Related Protein - genetics; Recombinant parathyroid hormone related protein; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Sea Bream; Sequence Alignment; Sparus aurata; Teleost
• ISSN: 0016-6480; 1095-6840
• DOI: 10.1016/j.ygcen.2005.02.020

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 1 mM isopropyl-β- d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1–125) as judged by SDS–PAGE and yielded up to 40 mg/L of culture medium (3.3 mg 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 (1–34 aa) PTHrP, the recombinant sbPTHrP(1–125) was equipotent with PTHrP(1–34) in displacing labelled 125I-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.