Prepn of recombinant human parathyroid hormone PTH (1-34)

A parathyroid hormone, crude technology, applied in the direction of parathyroid hormone, hormone peptide, recombinant DNA technology, etc., can solve the problems of high cost, unfavorable large-scale production, expensive enterokinase, etc., and achieve the relief of osteoporosis disease effect

Inactive Publication Date: 2003-05-14
西南生物工程产业化中试基地有限公司
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Problems solved by technology

However, enterokinase is expensive, making the proce

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  • Prepn of recombinant human parathyroid hormone PTH (1-34)
  • Prepn of recombinant human parathyroid hormone PTH (1-34)

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Embodiment 2

[0018] Example 2: Purification of PTH According to the method of the present invention, PTH expressing bacteria were constructed, cultured by fermentation, and collected by centrifugation. The bacterial cells were suspended in lysate (50mM Tris, 150mM NaCl, pH8.0), sonicated, and diluted and centrifuged to collect the inclusion bodies. It was washed with lysate and centrifuged to further purify PTH inclusion bodies. 20 g of inclusion bodies were suspended in 50 ml of 50 mM Tris, 150 mM NaCl (pH 8.0), 36 g of solid urea was slowly added under electromagnetic stirring to a final concentration of 6 M, and stirring was continued for 4 hours. Centrifugation to remove insoluble materials, 450ml of the supernatant was diluted with 50mM Tris, 150mM NaCl (pH8.0) until the concentration of urea was reduced to 1M and renatured overnight. The renatured solution was centrifuged to remove contaminated proteins, and the protein content in the supernatant was determined. Thrombin was added at an ...

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Abstract

The present invention relates to a preparation process of recombinant human parathyroid hormone PTH(1-34). The process constitutes a new engineering strain to result in high expression amount, simplepurifying course and low cost.The present invention adopts colibacillus degenerate codon modifying sequence corresponding cDNA, the basic sequence is L-V-P-R-PTH(1-34), where L-V-P-R is the enzyme incision site of thrombin. To the end of the nucleotides sequence, colibacillus terminator codon TAA is added; and in order to clone easily to the 5'-terminal enzyme incision site of EcoR I is added, and to the 3'-terminal, enzyme incision site of Sal I is added. High-purity recombinant huamn parathyroid hormone PTH(1-34) is obtained through fermentation of the engineering bacteria, occlusion body extraction, diluting renaturation, thrombase incision, anionic exchange chromatography and other steps.

Description

Technical field: [0001] The present invention relates to a method for preparing human parathyroid hormone PTH (1-34), in particular to a method for preparing the product using gene recombination technology, and the product can be used to treat osteoporosis. Background technique: [0002] Osteoporosis is a type of disease related to bone metabolism. It is a disease of non-specific bone metabolism disorder, which mainly occurs in the spongy bone part of the bone. Bone metabolism includes two aspects: osteoblast-mediated bone formation and osteoclast-mediated bone lysis and resorption. Bone metabolism is a dynamic equilibrium process of osteoblast-mediated bone formation and osteoclast-mediated bone lysis and resorption. [0003] With the increase of age, bone resorption activity exceeds bone formation activity, resulting in bone loss and bone density gradually decreasing. It is the phenomenon that bones appear hollow and loose, fragile and easy to fracture, which is called osteopor...

Claims

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Application Information

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IPC IPC(8): C07K14/635C12N1/21C12N15/63C12N15/64C12P21/02
Inventor 胡昌华李明何建军徐辉
Owner 西南生物工程产业化中试基地有限公司
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