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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]

Goodpasture antigen: Expression of the full-length α3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain. Background. Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the α3(IV) chain of type IV collagen, one of five α(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the α3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain. A knowledge of GP epitopes is important for the understanding of the etiology and pathogenesis of the disease and for the development of therapeutic strategies. Methods. A cDNA construct was prepared for the full-length human α3(IV) chain. The construct was stably transfected into human embryonic kidney 293 cells. The purified full-length r-α3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for binding of GP antibodies from five patients was compared with that of the human r-α3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay. Results. The r-α3(IV) chain was secreted from 293 cells as a single polypeptide chain that did not spontaneously undergo assembly into a triple-helical molecule. An analysis of GP-antibody binding to the full-length r-α3(IV) chain showed binding exclusively to the globular NC1 domain. Conclusion. The full-length human α3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontriple-helical NC1 domain of the α3(IV) chain, indicating the presence of specific immunogenic properties. The α3(IV) chain alone does not spontaneously undergo assembly... [ABSTRACT FROM AUTHOR]

Background: Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence. Methods: To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin. Of the 56 patients who had undergone renal transplantation, 33 had recurrences. Sixty-four patients, many of whom had undergone transplantation, were being treated with dialysis. Thirty-one patients with other renal diseases and nine normal subjects were also studied. Results: The 33 patients with recurrent focal segmental glomerulosclerosis after transplantation had a higher mean (±SE) value for permeability to albumin (0.47±0.06) than the normal subjects (0.06±0.07) or the patients who did not have recurrences (0.14±0.06). After plasmapheresis in six patients with recurrences, the permeability was reduced (from 0.79±0.06 to 0.10±0.05, P = 0.008), and proteinuria was significantly decreased. Patients with corticosteroid-sensitive nephrotic syndrome or with membranous nephropathy after transplantation had low levels of serum activity. The circulating factor bound to protein A and hydrophobic-interaction columns and had an apparent molecular mass of about 50 kd. Conclusions: A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury. (N Engl J Med 1996;334:878-83.) [ABSTRACT FROM AUTHOR]