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System and method for expressing and purifying recombinant protein with 3'-nucleotidase as tag and application of recombinant protein

A nucleotidase and recombinant protein technology, which is applied in the field of recombinant protein expression and purification systems, can solve the problems of low protein purity, prolong the sample loading time, reduce the sample loading speed, etc., so that the purification system is easy to operate and shortens the required time. , the effect of fast elution

Active Publication Date: 2015-01-21
海宁市袁花镇工业投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the purification system of maltose-binding protein are mainly the high price of the chromatographic medium and the ineffective binding of some recombinant proteins to the chromatographic medium, and the purity of the final protein rarely meets the purity requirements of structural biology (Austin, B. P., Nallamsetty, S. , and Waugh, D. S. (2009) Methods Mol Biol498, 157-172)
There are three main disadvantages of glutathione transferase as a label: GST is a homodimer and is not suitable for purifying multimeric proteins; its cysteine ​​oxidation can easily lead to protein aggregation and precipitation; when a large amount of crude extract needs When loading the column, the combination of GST and GSH gel is slow, so the loading speed must be reduced, and the prolonging of the loading time reduces the work efficiency (Arnau, J., Lauritzen, C., Petersen, G. E., and Pedersen, J. (2006 ) Protein expression and purification48, 1-13; Waugh, D. S. (2005) Trends in biotechnology23, 316-320; Hunt, I. (2005) Protein expression and purification40, 1-22)

Method used

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  • System and method for expressing and purifying recombinant protein with 3'-nucleotidase as tag and application of recombinant protein
  • System and method for expressing and purifying recombinant protein with 3'-nucleotidase as tag and application of recombinant protein
  • System and method for expressing and purifying recombinant protein with 3'-nucleotidase as tag and application of recombinant protein

Examples

Experimental program
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Embodiment 1

[0065] In this example, the protein expression and purification system was used to purify and analyze Escherichia coli APSK protein, and the kinetic analysis of the enzyme was carried out. The specific operation is as follows:

[0066] 1) Clone the yeast 3'-nucleotidase nucleotide sequence by PCR method (forward primer: GGAATTCCATATGCACCACCACCACCACGCATTGGAAAGAGAATTATTGG, reverse primer: AAGCTT CAGGGGCCCCTGGAACAGAACTTCCAG GGCGTTTCTTGACTGAATGAC, where the underline encodes the human rhinovirus 3C protease recognition sequence), and a human rhinovirus 3C protease recognition site was added to its C-terminus by PCR primer design.

[0067] 2) The 3'-nucleotidase nucleotide sequence and pET24a were subjected to restriction endonuclease Nde I / Hind After III double digestion, use T4 ligase to connect and construct the expression vector pND, the subsequent Hind III, not I, xho IMultiple cloning sites can be used to insert target protein gene sequences. The expressed rec...

Embodiment 2

[0078] Such as Figure 12 As shown, the nucleotide sequence of crucian carp leptin protein (forward primer: AAGCTTTATTTTCCAGCTCTTCTCTACCCATG; reverse primer: CCGCTCGAGTTAGCAGCTTTTCAACTGGTCC) was cloned by PCR method and the expression vector pND was digested with restriction endonuclease Hind III / Xho I respectively, and then used The crucian carp leptin protein expression vector pND-CCL was constructed by T4 ligase ligation.

[0079] Other technical features of this embodiment are as described in Embodiment 1.

Embodiment 3

[0081] Such as Figure 13 As shown, the nucleotide sequence of Guanxi honey pomelo ζ-carotene desaturase (forward primer: GCGGCCGCATGGGTTTCTTCAGTTCTGTTTC; reverse primer: CCGCTCGAGTTAGTTCATCGTTAGTAGTAGTTG) nucleotide sequence and expression vector pND were cloned by PCR method, and the restriction enzymes Not I / Xho After I double enzyme digestion, T4 ligase was used to construct Guanxi honey pomelo ζ-carotene desaturase expression vector pND-CPZDS.

[0082] Other technical features of this embodiment are as described in Embodiment 1.

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Abstract

The invention relates to the technical field of biology, in particular to a system and a method for expressing and purifying recombinant protein with 3'-nucleotidase as a tag and application of the recombinant protein. The system is characterized in by comprising a 3'-nucleotidase nucleotide sequence and a protein over-expression vector, a 3'-nucleotidase nucleotide sequence added with a protease recognition site, a protease nucleotide sequence matched with the protease recognition site, a lysis solution containing at least one of Li<+> and Ca<2+> ions, an eluent of a chelating agent containing at least one of Li<+> and Ca<2+> ions, and PAP (Prostatic Acid Phosphatase) agarose gel. The system disclosed by the invention is a simple, high-efficiency and economic system for purifying a target recombinant protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 3'-nucleotidase (3'- n Ucleoti d ase, 3-ND, EC 3.1.3.7) as a tagged recombinant protein expression and purification system, method and application. Background technique [0002] The structure and function research of proteins and polypeptides is particularly important in the post-genome era. There are many methods for protein purification, such as salting out and precipitation according to different solubility, ultrafiltration and gel filtration according to protein molecular size, electrophoresis and ion exchange chromatography according to protein charging properties, and the use of ligands to purify proteins. Affinity chromatography for specific binding, etc. Affinity chromatography covalently binds the ligand molecule to the solid support. When the protein mixture passes through the solid medium, the target protein can bind to the ligand and stay in the chromatography column...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66C07K1/16C07K14/245C07K14/46C12N9/02
Inventor 孙梅好杨依林杨洋王艳兴李鸿艳马建辉
Owner 海宁市袁花镇工业投资有限公司
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