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Recombination expression carrier and method for soluble expressing human I-type metallothionin

A metallothionein and expression vector technology, which is applied in the field of expressing and producing human type I metallothionein in a soluble form, and a recombinant expression vector for expressing human type I metallothionein, can solve the problem of expensive protease cost, time-consuming, and inability to obtain MT production and other issues

Active Publication Date: 2008-03-19
BIOPHARM RES & DEV CENT JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the solubility and yield of the protein have been improved (up to 8mg / L), time-consuming, expensive protease costs and the risk of protease hydrolysis of the target protein often fail to obtain satisfactory MT yields (see Hong et al., Protein Expression and Purification 2001, 21: 243-250)

Method used

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  • Recombination expression carrier and method for soluble expressing human I-type metallothionin
  • Recombination expression carrier and method for soluble expressing human I-type metallothionin
  • Recombination expression carrier and method for soluble expressing human I-type metallothionin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Artificial synthesis of small molecule ubiquitin-related modifier mature peptide and human type I metallothionein fusion protein gene

[0123] Thirteen oligonucleotide chains must be synthesized (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) , respectively: SUMO-F1 (SEQ ID No: 11), SUMO-F2 (SEQ ID No: 12), SUMO-F3 (SEQ ID No: 13), SUMO-F4 (SEQ ID No: 14), SUMO- R1 (SEQ ID No: 15), SUMO-R2 (SEQ ID No: 16), SUMO-R3 (SEQ ID No: 17), SUMO-R4 (SEQ ID No: 18), MT-F1 (SEQ ID No: 19), MT-F2 (SEQ ID No: 20), MT-R1 (SEQ ID No: 21), MT-R2 (SEQ ID No: 22) and MT-R3 (SEQ ID No: 23). The specific process of synthesis is shown in Figure 3. Briefly, first, in the first step of PCR reaction, 2 μl (10 μmol L -1 ), dNTP (each 2.5μmol·L -1 ) 10 μl, 10 μl of 10x PCR pfu buffer, add water to a total volume of 100 μl. After denaturation at 94°C for 5 minutes, slowly lower the temperature to 55°C, then add pfuDNA polymerase, then extend at 72°C for...

Embodiment 2

[0124] Embodiment 2: Construction of recombinant vector pET-Ulp1

[0125] First synthesize 18 oligonucleotide chains (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.), respectively: Ulp-F1 (SEQ ID No: 32), Ulp-F2 (SEQ ID No: 33), Ulp -F3 (SEQ ID No: 34), Ulp-F4 (SEQ ID No: 35), Ulp-F5 (SEQ ID No: 36), Ulp-F6 (SEQ ID No: 37), Ulp-F7 (SEQ ID No : 38), Ulp-F8 (SEQ ID No: 39), Ulp-F9 (SEQ ID No: 40), Ulp-R1 (SEQ ID No: 41), Ulp-R2 (SEQ ID No: 42), Ulp-R3 (SEQ ID No: 43), Ulp-R4 (SEQ ID No: 44), Ulp-R5 (SEQ ID No: 45), Ulp-R6 (SEQ ID No: 46), Ulp-R7 (SEQ ID No: 47 ), Ulp-R8 (SEQ ID No: 48) and Ulp-R9 (SEQ ID No: 49). According to the method described in Example 1 and the steps in Figure 4, three large fragments were artificially synthesized using PCR technology, named respectively as UI (SEQ ID No: 50), UII (SEQ ID No: 51) and UIII (SEQ ID No: 52). Then, using UI and UII as common templates, adding Ulp-F6 and Ulp-R3 as upstream and downstream primers,...

Embodiment 3

[0126] Embodiment 3: Construction of recombinant expression vector pET-SMT-Ulp1

[0127] In order to construct a cDNA sequence containing a mature peptide encoding a small molecule ubiquitin-related modification factor under the control of the T7 promoter and a human type I metallothionein fusion protein cDNA sequence connected in tandem under the control of the T7 promoter and under the control of the T7 promoter The recombinant expression vector of the ubiquitin-related modifier protease 1 gene, first, synthesize an oligonucleotide primer Ulp1-T7-F based on the T7 promoter: 5'CT GGA TCC GAA TTC GAG CTC AAG CTT CTC GAGTAA TAC GAC TCA CTA TAG GGA G3' (synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.; SEQ ID NO: 54). The 5' end of this primer contains a multiple cloning site. According to the conventional PCR method, use the recombinant expression vector pET-Ulp1 as the template, Ulp1-T7-F and Ulp-R9 as the upstream and downstream primers, and enter t...

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Abstract

The present invention relates to a method for producing a human I type metal sulfur protein (Human metallothionein-I, hMT-I), in particular to the fusion expression by adopting the small molecule ubinquitin-related modifier mature peptide (Small Ubiquitin-related Modifier, SUMO) and hMT-I, and the fusion protein and the ubinquitin-related modifier protease 1 (Ubiquitin-like protease 1) are co-expressed in a prokaryotic organism. In the fermentation and expression process, the ubiquitin associative modifier gene protease 1 can hydrolyze the fusion protein which consists of the ubinquitin-related modifier mature peptide and the hMT-I, to produce the dissoluble hMT-I.

Description

technical field [0001] The invention belongs to the field of genetic engineering. More specifically, the present invention relates to a recombinant expression vector for expressing human type I metallothionein and a method for expressing and producing human type I metallothionein in a soluble form. Background technique [0002] Metallothionein (MT) is a class of low molecular weight, cysteine-rich, metal-binding proteins that can bind heavy metals and be induced by metals. Since metallothionein was first isolated in 1957, researchers have found that MT widely exists in the tissues of animals, plants and eukaryotic microorganisms. [0003] There are 4 MT subtypes in mammals, namely MT-I, MT-II, MT-III, MT-IV, where MT-I and MT-II are present in all organs and MT-III is only present in brain tissue , while MT-IV is only present in squamous epithelial tissues (Uchida et al., J Biol. Chem., 1995, 270: 3365-3369). Typical mammalian MT has (1) low molecular weight, generally on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/62C07K14/435C07K19/00
Inventor 黄亚东刘锦棠苏志坚肖巧学柳忠玉丁长才冯娅
Owner BIOPHARM RES & DEV CENT JINAN
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