Evaluation of a New Method for Biological Activity Analysis of Recombinant Human Parathyroid Hormone-Related Protein

Abstract

Background: BaParathyroid hormone-related protein (PTHrP) is a multi-functional protein with a sequence “aa 1-34” similar to that of parathyroid hormone (PTH). In spite of growth factor activity, there is no standardized analytical method to determine the biological activity of recombinant PTHrP. Methods: The current study isolated the cDNA encoding 141 N-terminal amino acid sequence of PTHrP, cloned it in plasmid vec-tor pET32a, and produced recombinant PTHrP in Escherichia coli . The biological activity analysis of the recombinant protein was evaluated using a precise and practical method that measured PTHrP-mediated cell proliferation of a breast cancer cell line MCF7. Additionally, to investigate the accuracy and precision of MCF-7 method used, chondrogenic differentiation condition of PTHrP treated mesenchymal stem cells (MSCs) was evaluated. Results: DNAsequencingand protein massspectrometryanalysisconfirmedtheaccuracy of clonedcDNAsequenceandexpressed the protein. Biological activity analysis showed the increasing effect of recombinant PTHrP on cell proliferation of cancer cell line MCF-7. Alkaline phosphatase and gene expression analysis validated the anti-hypertrophy effects of recombinant PTHrP on chondrogenic differentiation of MSCs. Conclusions: The current study method removed potential challenges and errors in comparability and reproducibility studies focusing on stem cell differentiation and pathogenesis of breast cancer arising from PTHrP biological activity analysis.