Double-label vector for expression, refolding and modification of long-acting protein medicament and method

A protein and double tag technology, applied in the field of genetic engineering, can solve the problems of efficient and difficult to take into account of proteins, and achieve the effect of promoting construction

Inactive Publication Date: 2015-01-07
程永升 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the introduction of additional amino acids cannot be effective for all proteins, and it is difficult to take into account that it does not affect the activity of the protein drug itself and can be chemically modified conveniently.

Method used

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  • Double-label vector for expression, refolding and modification of long-acting protein medicament and method
  • Double-label vector for expression, refolding and modification of long-acting protein medicament and method
  • Double-label vector for expression, refolding and modification of long-acting protein medicament and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1: Construction of pSumo-Intein plasmid vector

[0082] (1) Obtain the humanized Sumo3 protein sequence (P55854) through the Uniprot database, and then use the protein sequence reverse translation and codon optimization service (http: / / genomes.urv.es / OPTIMIZER / ) to obtain the Sumo3 DNA sequence ( SEQ ID No. 1). On this basis, its 7-276 bp was taken as the sequence of sumo3tag (SEQ ID No.2), and this sequence also contained a little 6×His tag sequence to facilitate IMAC affinity denaturation and purification later.

[0083] (2) Next, design segmented primers (Gene Design, http: / / 54.235.254.95 / gd / ) for gene synthesis on the optimized DNA sequence:

[0084] The synthesis reaction is accomplished by Assembly PCR reaction (Marchand, J.A., and Peccoud, J. (2012). Building block synthesis using the polymerase chain assembly method. Methods Mol. Biol. 852, 3–10.). The sequences OPM and TPM of the segmented primer are shown in SEQ ID NO.6-15. All designed Overlap p...

Embodiment 2

[0088] Embodiment 2: Preparation of human source SENP2 catalytic domain protein

[0089] (1) Obtain the human SENP2 protein sequence (Q9POU3) through the Uniprot database, and then use the protein sequence reverse translation and codon optimization service (http: / / genomes.urv.es / OPTIMIZER / ) to obtain the SENP2 catalytic domain (362-589aa ) DNA sequence (SEQ ID No.3);

[0090] (2) Next, design segmented primers (Gene Design, http: / / 54.235.254.95 / gd / ) for gene synthesis on the optimized DNA sequence:

[0091] The sequences OPM and TPM of the segmented primer are shown in SEQ ID NO.26-43. The synthesis reaction is accomplished by Assembly PCR reaction (Marchand, J.A., and Peccoud, J. (2012). Building block synthesis using the polymerase chain assembly method. Methods Mol. Biol. 852, 3–10.). All designed Overlap primers (ie segmented primers) were synthesized by Sigma and dissolved in aqueous solution to form a 100uM stock solution. Prepare template primer mix (TPM) (SEQ ID NO....

Embodiment 3

[0099] Example 3: Plasmid Construction of Proinsulin and GLP1 Analogs in pSumo-Intein System

[0100] (1), construction of pSumo-proinsulin-Intein plasmid vector

[0101] a. Obtain the sequence of human Proinsulin protein (P01308) through the Uniprot database, and then obtain the prokaryotic expression mutant through the program. The sequence of the mutant is separated by using KR as the linker sequence between the B chain and A chain sequences of Insulin, Form the structure of B chain-KR-A chain; then use protein sequence reverse translation and codon optimization service (http: / / genomes.urv.es / OPTIMIZER / ) to obtain its DNA sequence (SEQ ID No.4).

[0102] b. Design segmented primers (Gene Design, http: / / 54.235.254.95 / gd / ) for the above-mentioned optimized DNA sequence for gene synthesis:

[0103] The synthesis reaction was accomplished by Assembly PCR (Marchand, J.A., and Peccoud, J. (2012). Building block synthesis using the polymerase chain assembly method. Methods Mol. B...

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Abstract

The invention belongs to the field of genetic engineering and provides a double-label vector for expression, refolding and modification of a long-acting protein medicament and a method. The method comprises the following steps: constructing a pSumo-Intein plasmid vector, preparing a human SENP2 catalytic domain protein, constructing a pSumo-target DNA-Intein plasmid vector, expressing, refolding and purifying the pSumo-target DNA-Intein plasmid vector in expression host bacteria and the like to obtain a target protein. According to the double-label vector provided by the invention, two labels, namely Sumo and Intein are integrated into a same expression plasmid, and the advantages of the two labels can be simultaneously utilized to prevent degradation by a protease to the greatest extent during expression in colon bacillus. The double-label vector provided by the invention can be used for large-scale and low-cost implementation of purification and refolding of the protein medicament from the laboratory scale to the industrial production scale, high-throughput protein antigen expression and long-acting transformation of the existing protein medicaments.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a double-label carrier and method for expressing and refolding modification of long-acting protein drugs. Background technique [0002] Since the invention of gene recombination technology (Hannig, G., and Makrides, S.C. (1998). Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol. 16, 54–60.), recombinant protein peptide drugs have gradually replaced traditional sources Advanced protein drugs, such as human recombinant insulin, have basically completely replaced insulin from animal pancreas to treat diabetes (EP2374888A1, EP0437320A1). At present, the market for recombinant protein drugs has reached 140 billion US dollars worldwide, and with the discovery of antibody drugs and other new protein drugs, this market has continued to maintain a high growth rate for a long period of time. [0003] The production of recombinan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/62C07K14/575
Inventor 程永升吴雪萍
Owner 程永升
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