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SOD-ELP fusion protein and preparation method thereof

A fusion protein and elastin-like technology, which is applied in the field of purification of superoxide dismutase, SOD-ELP fusion protein and its preparation, can solve the problems of complicated purification process, low expression of target protein, unpredictable activity of fusion protease, etc. , to achieve the effect of low immunogenicity, prolonging half-life, and easy degradation by the body

Pending Publication Date: 2020-04-17
NANJING UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are precedents for the production of human superoxide dismutase by genetic engineering technology, but there are still many problems: mainly: (1) the expression level of the target protein is not high (2) the purification process is complicated, requiring ion exchange, molecular Purification by a series of chromatographic methods such as size exclusion
The fusion protein of ELPs and SOD has not been reported yet, and it is still unpredictable whether the ELPs involved in the fusion with SOD can maintain the reversible phase transition properties of ELPs, and be used for the expression and purification of SOD, and whether the fusion protein has enzymatic activity.

Method used

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  • SOD-ELP fusion protein and preparation method thereof
  • SOD-ELP fusion protein and preparation method thereof
  • SOD-ELP fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1 Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the ELP[H33V7]40 gene in advance (see SEQID NO.2 for sequence information) and pre-insert it between BamHI and XhoI of the pET28a vector to form pET28a-ELP 40 carrier.

[0060] 2. Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize SOD gene (see SEQ ID NO.3 for sequence information) (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) and PCR primers ( For the sequence information, see SEQ ID NO.6 and SEQ ID NO.7), the PCR conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 25 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 25 seconds, 30 cycles, extension at 72°C for 10 minutes, and 10 ℃ insulation.

[0061] 3. The PCR product of SOD gene was mixed with pET28a-ELP respectively 40 The vector was digested with NdeI and BamHI and purified with the kit, and the PCR digestion product of SOD and pET28a-ELP containing ELP[H33V7]40 ...

Embodiment 2

[0065] 1 Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the ELP[V10G6A4]20 gene in advance (see SEQ ID NO.1 for sequence information) and pre-insert it between BamHI and XhoI of the pET28a vector to form pET28a-ELP 20 carrier.

[0066]2. Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize PCR primers for SOD (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) (see SEQ ID NO.6 and SEQ ID NO for sequence information .7), the PCR conditions are 95°C pre-denaturation for 5min, 95°C denaturation for 25s, 56°C annealing for 30s, 72°C extension for 25s, 30 cycles, 72°C extension for 10min, and 10°C incubation.

[0067] 3. Combine the PCR product of SOD gene with pET28a-ELP containing ELP[V10G6A4]20 synthesized by commission 20 The vector was digested with NdeI and BamHI and purified with the kit, respectively digested and ligated according to the instructions of the M2200S Quick ligation kit of NEB Company, and introduced into DH5α ...

Embodiment 3

[0071] Example 3 fusion protein ELP 40 - Construction and expression of SOD

[0072] In order to compare the fusion protein of SOD and elastin-like polypeptide in two different ways, that is, SOD-ELP 40 and ELP 40 -Differences in the expression of SOD, the vector pET28a-ELP was constructed 40 -SOD.

[0073] 1. Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize PCR primers for SOD (Cu, Zn Superoxide Dismutase, Cu, Zn-SOD, EC 1.15.1.1, GenBank: AY049787) (see SEQ ID NO.6 and SEQ ID NO for sequence information .7), the PCR conditions are 95°C pre-denaturation for 5min, 95°C denaturation for 25s, 56°C annealing for 30s, 72°C extension for 25s, 30 cycles, 72°C extension for 10min, and 10°C incubation. The PCR product was pre-inserted between BamHI and XhoI of the pET28a vector to form a pET28a-SOD vector.

[0074] 2. Entrust Nanjing GenScript Biotechnology Co., Ltd. to synthesize the ELP[H33V7]40 gene in advance (see SEQ ID NO.2 for the sequence information). Ent...

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Abstract

The invention discloses an SOD-ELP fusion protein and a preparation method thereof. The SOD-ELP fusion protein is composed of ELP20 or ELP40 and SOD, the SOD-ELP40 fusion protein is purified by adopting three rounds of ITC, so that the purification process of the protein is simple, economic and efficient, and the method can be used for industrially preparing the recombinant humanized copper-zinc superoxide dismutase with high specific activity on a large scale. The fusion protein not only retains the activity of the enzyme, but also has good heat resistance and biocompatibility, and greatly prolongs the simulated plasma half-life period of the enzyme. According to the invention, the purified SOD-ELP40 is further embedded into a lipidosome, so that a lipidosome with high encapsulation efficiency and a transdermal effect is obtained. The SOD-ELP40 prepared by the invention can be widely applied to the fields of cosmetics, foods, medicines and the like.

Description

technical field [0001] The invention belongs to the field of protein engineering, and relates to a SOD-ELP fusion protein and a preparation method thereof, in particular to a method for purifying superoxide dismutase through a non-chromatographic method by utilizing the elastin-like phase transition property. Background technique [0002] Superoxide dismutase (Superoxide Dismutase, referred to as SOD), as an active substance widely present in living organisms, can remove harmful substances produced during the metabolic process of animals, plants and microorganisms. In 1938, scientists discovered superoxide dismutase from bovine red blood cells, and the research on superoxide dismutase has been going on for 80 years. [0003] SOD is a kind of metalloenzyme that widely exists in the biological world. It can catalyze the disproportionation reaction of superoxide anion radicals and balance the functions of oxygen free radicals in the body. It is the key line of defense for organ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C07K19/00C12R1/19
CPCC12N15/70C12N9/0089C12Y115/01001C07K14/00C07K2319/00
Inventor 樊燕蓉陈星宇冯娇燕周奔赵广义徐根兴
Owner NANJING UNIV OF SCI & TECH
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