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

Gen Comp Endocrinol. 2005 Aug;143(1):57-65. doi: 10.1016/j.ygcen.2005.02.020. Epub 2005 Apr 2.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Cyclic AMP / metabolism*
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Vectors
  • Molecular Sequence Data
  • Parathyroid Hormone-Related Protein / biosynthesis*
  • Parathyroid Hormone-Related Protein / genetics*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Sea Bream*
  • Sequence Alignment


  • DNA, Complementary
  • Parathyroid Hormone-Related Protein
  • Recombinant Proteins
  • Cyclic AMP