Miraculin recombinant protein and expression and purification method thereof

A technology of recombinant protein and miraculin, which is applied in the field of genetic engineering to achieve the effect of simple operation and high protein purity

Pending Publication Date: 2021-02-02
珠海中科先进技术研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For the expression and purification of miraculin, no specific studies have shown which carrier is more suitable

Method used

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  • Miraculin recombinant protein and expression and purification method thereof
  • Miraculin recombinant protein and expression and purification method thereof
  • Miraculin recombinant protein and expression and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Optimize the miraculin gene codon and synthesize the miraculin gene sequence

[0036] Because Escherichia coli has a preference for amino acid codons, the translation efficiency of different codons for the same amino acid is different in Escherichia coli, in order to improve the expression level of miraculin as the target protein in Escherichia coli, according to the mysterious The coding gene sequence of fruit element in NCBI (GenBank Accession No.D38598) optimizes the codons of amino acids encoded by the target protein.

[0037] The amino acid sequence of miraculin recombinant protein is shown in SEQ ID NO: 1:

[0038]Met Lys Glu Leu Thr Met Leu Ser Leu Ser Phe Phe Phe Val Ser Ala LeuLeu Ala Ala Ala Ala Asn Pro Leu Leu Ser Ala Ala Asp Ser Ala Pro Asn Pro ValLeu Asp Ile Asp Gly Glu Lys Leu Arg Thr Gly Thr Asn Tyr Tyr Ile Val Pro ValLeu Arg Asp His Gly Gly Gly Leu Thr Val Ser Ala Thr Thr Pro Asn Gly Thr PheVal Cys Pro Pro Arg Val Val Gln Thr Arg Lys Glu Val Asp His Asp...

Embodiment 2

[0041] Verification experiment on the expression and purification of miraculin recombinant protein expression vectors with different tags at the carboxy-terminus

[0042] In this embodiment, the expression vectors of miraculin (miraculin) recombinant protein with His tag at the carboxyl end and the expression vector of miraculin (miraculin) recombinant protein with FLAG tag at the carboxyl end were respectively constructed, and respectively Transformed into Escherichia coli DH5α competent cells to preliminarily screen the correct expression vector, and then respectively transformed into genetically engineered bacteria Escherichia coli Origami (DE3) to investigate the expression of miraculin recombinant protein.

[0043] The plasmid used in this example is pUC57-miraculin, and the specific synthesis method is to artificially synthesize miraculin recombinant protein containing restriction site BamHI (GGATCC) at the 5' end and Hind III (AAGCTT) at the 3' end The gene of the mirac...

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Abstract

The invention relates to a miraculin recombinant protein and an expression and purification method thereof. The carboxyl terminal of the miraculin recombinant protein expression vector used in the method is provided with a FLAG label, and Escherichia coli Origami (DE3) is adopted, so that a large amount of miraculin recombinant protein can be rapidly obtained, and a new method is provided for solving the problem of miraculin source and yield limitation, meanwhile, rich raw materials are provided for developing a new sweetening agent and researching a taste changing mechanism of the miraculin.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a miraculin recombinant protein and its expression and purification method. Background technique [0002] Low-calorie sweeteners become the new pursuit; artificial sweeteners such as: saccharin, aspartame, cyclamic acid and acetaminophen, acesulfame-K may cause various diseases such as: mental health problems, mental illness , bladder cancer, heart failure and brain tumors; natural sweet proteins are the new favourites. [0003] Miracle fruit, whose Latin scientific name is Synsepalum dulcificum Daniell, also known as dream fruit, miracle fruit, West African sapota, Mira holy fruit, is a typical sapotaceae miracle fruit plant, a tropical evergreen shrub. Miraculin is a basic glycoprotein naturally present in the pulp of miracle fruit, which can make the sour taste sweet at very low concentrations. Miraculin is a homodimeric sugar composed of a single polypeptide An ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/70C12N15/66C12N15/62C07K19/00C07K1/14
CPCC07K14/415C12N15/70C12N15/66C07K2319/43
Inventor 卢申姣徐畅谢桦函陈婉玲高飞姜长安
Owner 珠海中科先进技术研究院有限公司
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