Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139)

Cai Wu, Patricia K. Seitz, Miriam Falzon

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Recombinant human parathyroid hormone-related protein (hPTHrP) (1-139) was expressed using the IMPACT T7 (intein-mediated purification with an affinity chitin-binding tag) system, allowing purification of free recombinant peptide in a single chromatographic step. This system utilizes an intein, which is a protein splicing element from the Saccharomyces cerevisiae VMA1 gene. The intein has been modified so that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of 1,4-dithiothreitol (DTT). The cDNA encoding hPTHrP (1-139) was cloned into the pTYB1 vector to create an in-frame fusion at the N-terminus of the intein gene. The cDNA for the chitin-binding domain from Bacillus circulans is present at the C-terminus of intein for affinity purification of the three-part fusion protein on a chitin column. The recombinant plasmid was transfected into E. coli ER2566 cells and synthesis of the PTHrP fusion protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG). This system produced pure hPTHrP (1-139) and an N-terminally truncated analogue, hPTHrP (27-139), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis, N-terminal sequence analysis and mass spectroscopy. hPTHrP (1-139) stimulated cAMP accumulation in ROS 17/2.8 osteoblastic bone cells, whereas hPTHrP (27-139) failed to elicit a response. hPTHrP (1-139) also inhibited the growth of the breast cancer cell line MDA-MB-231; the magnitude of the response was comparable with that of synthetic hPTHrP (1-34) and (1-86). Neutralization of endogenous PTHrP and added hPTHrP (1-139) and N-terminal species with an anti-PTHrP antiserum completely abolished the growth inhibitory effects. These results indicate that the added peptides modulate cell growth by acting at the cell surface. Availability of recombinant hPTHrP (1-139) will allow further study of its biological function, as well as its structure.

Original languageEnglish (US)
Pages (from-to)163-174
Number of pages12
JournalMolecular and Cellular Endocrinology
Volume170
Issue number1-2
DOIs
StatePublished - Dec 22 2000

Fingerprint

Distillation columns
Inteins
Parathyroid Hormone-Related Protein
Purification
Chitin
Fusion reactions
Growth
Complementary DNA
Genes
Protein Splicing
Thiogalactosides
Peptides
Proteins
Dithiothreitol
Cell growth
Bacilli
parathyroid hormone-related protein (1-139)
Electrophoresis
Sodium Dodecyl Sulfate
Yeast

Keywords

  • Breast cancer
  • Cell proliferation
  • IMPACT T7
  • Parathyroid hormone-related protein
  • Recombinant peptide

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1-139). / Wu, Cai; Seitz, Patricia K.; Falzon, Miriam.

In: Molecular and Cellular Endocrinology, Vol. 170, No. 1-2, 22.12.2000, p. 163-174.

Research output: Contribution to journalArticle

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