Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacterium

A parathyroid hormone and fusion protein technology, applied in the field of parathyroid hormone PTH fusion protein and expression vector, can solve the problems of difficulty in self-preparation, high price and the like, and achieve the advantages of few steps, high yield and increased economic benefits. Effect

Active Publication Date: 2020-03-31
重庆艾力彼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Factor Xa recognizes the site Ile-Glu / Asp-Gly-Arg↓, and enterokinase recognizes Asp-Asp-Asp-Asp-Lys↓. After cutting the fusion protein, no redundant amino acids will be left at the amino terminal of the target protein, but Both enzymes are expensive and not easy to prepare yourself

Method used

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  • Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacterium
  • Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacterium
  • Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construction and expression of embodiment 1 recombinant PTH (1-34) engineering bacteria

[0061] PTH(1-34) is the amino acid sequence of SEQ ID NO:9, and the encoding DNA code is optimized as SEQ ID NO:10.

[0062] 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.

[0063] 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.

[006...

Embodiment 2

[0069] Example 2 Establishment of PTH(1-34) Character Preliminary Confirmation Criteria

[0070] The 8th and 18th positions of PTH(1-34) are Met, which is easy to be oxidized, and the biological activity of oxidized PTH(1-34) is greatly reduced. Therefore, it is necessary to identify the fusion protein expressed by the strain initially screened and the target protein initially purified to eliminate substandard recombinant engineering bacteria.

[0071] The simple and easy standard is to use the retention time of the main peak of HPLC to judge, because PTH(1-34) will appear new peaks with different retention times under different degrees of oxidation. If the main peak retention time of the PTH (1-34) obtained by the expression and final purification of the screened recombinant engineered bacteria is not within 100±0.03%, then the recombinant engineered bacteria are considered as unqualified.

[0072] Therefore, in this embodiment, the PTH (1-34) standard of WHO is used to dete...

Embodiment 3

[0081] Example 3 Preliminary purification of fusion protein, purification of target protein PTH (1-34) and preliminary identification of its properties

[0082] Purify the expressed fusion protein with Ni-NTA according to the properties of the purification tag. For soluble expression, use the supernatant of broken bacteria to combine with the filler, and add urea at a final concentration of 8mol / L to dissolve the inclusion body and then combine with the filler. Wash the miscellaneous protein according to the washing steps recommended by the filler manufacturer, and then use the eluent to elute the fusion protein. . After diluting the eluate (after renaturation of the eluate from the inclusion body), combine with the corresponding filler, respectively use the corresponding protease to carry out enzymatic digestion on the filler, collect the flow-through, and use 10% SDS-PAGE and HPLC (comparative example 2) Preliminary identification of the properties of the target protein.

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Abstract

The invention discloses a fusion protein for expressing recombinant parathyroid hormone (PTH). The fusion protein is sequentially composed of an expression enhancing peptide, a connecting peptide 1, His6, a connecting peptide 2, a guide protein, a connecting peptide 3, a protease cleavage site and parathyroid hormone (PTH) (1-34). The invention also provides recombinant plasmids and engineering bacteria for expressing the fusion protein of the recombinant parathyroid hormone PTH. According to the invention, the fusion protein expressed by an approximate inclusion body and a stable recombinantengineering strain can be obtained. Through protease digestion of the expression protein and purification of a few steps, the target protein PTH (1-34) with purity higher than 99.5% and good biological activity can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and relates to a fusion protein and an expression carrier of parathyroid hormone PTH (1-34). Background technique [0002] Parathyroid hormone is synthesized by parathyroid chief cells and contains 84 amino acids. After parathyroid hormone is secreted into the blood, it exists in three forms. The first type is a complete 84 amino acids (PTH(1-84)); the second type contains amino acids 1-34 (PTH(1-34)); the third type contains amino acids 55-84 . Both PTH(1-84) and PTH(1-34) have complete biological activity to regulate blood calcium concentration in the body through target organs such as bones and kidneys. PTH(1-34) contains all the necessary structures to express all the biological activities of PTH, and has the biological activity of complete PTH; and it is better than PTH(1-84) in promoting the growth of osteoblasts and stimulating bone formation. [0003] The preparation of PTH(1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12N1/21C12R1/19
CPCC07K14/635C12N15/70C07K2319/50C07K2319/02C07K2319/23C07K2319/24C07K2319/20
Inventor 冯强郭刚张欣罗莉吴翼卢彭封曾昭坤
Owner 重庆艾力彼生物科技有限公司
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