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Human parathyroid hormone (hPTH), synthesized in the parathyroid gland as a linear peptide, is one of the key regulatory molecules in calcium homeostasis and bone resorption. The N terminus region of hPTH (1-34) is a functionally important part of the molecule that is sufficient and necessary for potently executing most of the hormonal actions. Therefore, hPTH (1-34) is considered to be an attractive therapeutic agent in the treatment of osteoporosis. Here, we describe a high yield expression and purification method for the production of hPTH (1-34) from Escherichia coli. The hPTH (1-34) was expressed as a fusion protein in soluble form, and found to be more than 25% of total proteins. The fusion protein was first purified by GST affinity chromatography and, after cleavage of GST with Factor Xa, the peptide (hPTH 1-34) was further purified by reversed phase Source-30™ chromatography. This double purification strategy produced 30mg/l of hPTH (1-34) with purity = 98%. The identity of the purified peptide was confirmed by mass spectrometry and N-terminal sequencing analysis. The biological activity of the peptide was confirmed in the rat osteogenic cell line UMR-106 by measuring cAMP levels, which were identical to hPTH standards, indicating that purified rhPTH (1-34) has full biological activity. [ABSTRACT FROM AUTHOR]

Abstract: Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100μg/ml. This method yields greater than 90% of the dimer form with a yield of 40mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2×107 to 4×107 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule. [Copyright &y& Elsevier]