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Purification method of recombinant parathyroid hormone PTH (1-34)

A parathyroid hormone, separation and purification technology, applied in the field of separation and purification of recombinant parathyroid hormone PTH, can solve the problems of complex process flow, influence on polypeptide activity, low yield, etc., and achieve the effect of shortening purification process and simple operation

Active Publication Date: 2020-04-17
重庆艾力彼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods generally have problems such as the need to use organic solvents to affect the activity of the peptide, the peptide is easily degraded, the yield is low, and the process is complicated.

Method used

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  • Purification method of recombinant parathyroid hormone PTH (1-34)
  • Purification method of recombinant parathyroid hormone PTH (1-34)
  • Purification method of recombinant parathyroid hormone PTH (1-34)

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Construction and expression of embodiment 1 recombinant PTH (1-34) engineering bacteria

[0057] PTH(1-34) is the amino acid sequence of SEQ ID NO:1, and the encoding DNA code is optimized to SEQ ID NO:2.

[0058] Three segments of genes including TEV enzyme cleavage site (ENLYFQ), enterokinase enzyme site (DDDDK) and factor Xa cleavage site (IEGR) were designed at the N-terminus of PTH (1-34). Add (GGGGS)3, His6 and SSGSSG sequentially from right to left, add BamHI site GGATCC at the 5' end of each gene, add stop codon TGATAA, HindIII site AAGCTT, NotI site GCGGCCGC at the 3' end, and submit the sequence to Shanghai Synthesized by Bioengineering Ltd.

[0059] Respectively use BamHI+HindIII and BamHI+NotI to double-enzyme digest the synthesized three-segment gene and each vector as shown in Table 1, and recover the target fragment and vector respectively. The construction of the recombinant expression vector was carried out according to the manner in Table 1.

[0060...

Embodiment 2

[0064] Embodiment 2: the optimized construction of recombinant PTH (1-34) engineering bacterium

[0065] The preliminary results obtained by summarizing the fusion protein expressed by the recombinant vector constructed in Example 1 are shown in Table 2,

[0066] Table 2 The expression of each recombinant engineered bacteria

[0067]

[0068] If it is a soluble expression fusion protein, the obtained target protein PTH(1-34) is an oxidized form; if it is an inclusion body expression, the fusion protein needs to be fully refolded before protease digestion, and the existing purpose must be followed. In the old way of protein preparation, a reverse-phase purification step must be added to the purification process, and a large amount of organic solvents are used in the preparation process.

[0069] In order to overcome the disadvantages of the prior art, the optimal construction strategy of the present application for the recombinant PTH (1-34) engineering bacteria is: the rec...

Embodiment 3

[0071] Example 3 Bacteria Breakdown and Treatment of Inclusion Body

[0072] Bacteria resuspension: the pET28a-PTH(1-34) / BL21 Escherichia coli engineered bacteria were fermented at high density, and the bacteria were collected by centrifugation for later use. Take about 50g of bacteria, mix and suspend with PBS buffer solution (liquid A) of pH6.0-pH8.5 according to the weight (g): volume (ml) ratio of 1:5-10, and pre-cool at 4°C.

[0073] High-pressure sterilization: Use distilled water to flush the pipeline of the high-pressure homogenizer, and turn on the low-temperature circulation system to pre-cool to 1-4°C for later use. Add the pre-cooled suspended bacteria liquid to a high-pressure homogenizer, maintain the pressure at 60-80Mpa to break the bacteria 3-5 times, take a smear of the broken bacteria liquid and stain it with crystal violet, and the unbroken bacteria in each field of view of the oil immersion lens is less than 1-2. For the destruction of bacteria completely...

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Abstract

The invention discloses a separation and purification method of recombinant parathyroid hormone (PTH), which comprises the following steps: 1) bacterium crushing and inclusion body treatment: carryingout high-pressure homogenization and bacterium crushing, carrying out high-speed centrifugation after bacterium crushing to collect precipitates, carrying out resuspension washing on the precipitates, and mixing and redissolving the precipitates with a redissolved solution; 2) affinity chromatography; wherein a filler is selected from one of Ni-NTA, GST Sepharose and MBP Sepharose; 3) carrying out enzyme digestion and renaturation on a column; 4) carrying out cation exchange chromatography and / or hydrophobic chromatography, and (5) desalting. According to the present invention, through the method, the PTH (1-34) polypeptide with biological activity can be efficiently extracted from an inclusion body so as to make the large-scale industrial production of the PTH (1-34) possible.

Description

technical field [0001] The invention belongs to the technical field of protein separation and purification, and in particular relates to a method for separating and purifying recombinant parathyroid hormone PTH (1-34). Background technique [0002] Parathyroid hormone (PTH) is a polypeptide hormone composed of 84 amino acids, which is synthesized, stored and secreted by parathyroid epithelial cells. The PTH entering the blood circulation will quickly transform into two peptides at the amino-terminal and carboxy-terminal, among which the amino-terminal 1-34 amino acid residue peptide has complete PTH biological activity, regulates blood calcium concentration and bone metabolism, has little immune activity, and The peptide at the carboxyl end is a PTH immune active polypeptide, and its biological role is not very clear. [0003] PTH mainly exerts the biological function of anti-osteoporosis, and its influence on bone formation is mainly produced by acting on bone tissue cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/635C07K1/18C07K1/20C07K1/22
CPCC07K14/635Y02A50/30
Inventor 郭刚冯强张欣曾昭坤卢彭封卢文根熊峰杨念
Owner 重庆艾力彼生物科技有限公司
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