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

Abstract

Background: Parathyroid 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 vector 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 MCF-7. 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: DNA sequencing and protein mass spectrometry analysis confirmed the accuracy of cloned cDNA sequence and expressed 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 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.

Micro- and nanotechnology in biomedical engineering for cartilage tissue regeneration in osteoarthritis

“…Chondrogenic differentiation is a phased process with an expression of early and late chondrogenic markers. ALP and type-X collagen are the most well-known markers of hypertrophy, a natural phenomenon during the terminal differentiation of mature chondrocytes before bone formation [ 212 – 214 ]. Le et al have selected seven patterns from a library of previously created microtopography platforms, which induced high ALP expression and used them to analyze the chondrogenic differentiation markers of ATDC5 cells [ 215 ].…”

An osteoinductive surface by adhesive bone morphogenetic protein-2 prepared using the bioorthogonal approach for tight binding of titanium with bone

“…Nasrabadi et al 41 have reported the successful purification of recombinant BMP-2 using heparin-immobilized chromatography owing to the heparin-binding domain of BMP-2, whereas Gieseler et al 42 used cation exchange column chromatography. By using LC/MS-MS, we confirmed that all 8 tyrosine residues converted from Tyr to DOPA.…”

“…10,11,38 Although various approaches have been tested, the process of preparing biologically active BMP-2 dimers remains challenging. [39][40][41][42] The objective of the present study was to create an adhesive BMP-2 that can be stably adsorbed onto metals, such as Ti, to promote Ti binding to bones. We aimed to design a highly active adhesive and dimer-forming BMP-2 using bioorthogonal chemistry, wherein recombinant DNA technology was combined with enzymatic modifications to achieve long-term osseointegration with Ti.…”

“…10,11,38 Although various approaches have been tested, the process of preparing biologically active BMP-2 dimers remains challenging. 39–42…”