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High efficiency ELP fusion protease as well as preparation and application thereof

A fusion protein and protease technology, applied in the application field of cutting fusion protein, can solve the problem of high specificity of protease digestion

Inactive Publication Date: 2009-04-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, the conditions of protease cleavage are much milder than those of chemical methods, and protease has high specificity for cleavage, and there are many kinds of proteases to choose from, so the use of protease in fusion protein cleavage is the first choice, although commercial proteases are more expensive

Method used

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  • High efficiency ELP fusion protease as well as preparation and application thereof
  • High efficiency ELP fusion protease as well as preparation and application thereof
  • High efficiency ELP fusion protease as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of ELP-3C protease expression vector and transformation of host bacteria

[0032] Synthetic ELP gene (Meyer, D.E., and Chilkoti, A.1999.Purification of recombinant proteins by fusion with thermally-responsive polypeptides.NatBiotechnol 17:1112-1115.) according to the method reported by people such as Meyer and Chilkoti in 1999, its nucleoside The acid sequence is shown in SEQ ID NO.1. After being digested with EcorI at the N-terminus and HindIII at the C-terminus of the ELP gene, it was cloned into the corresponding restriction sites in the commercialized prokaryotic expression vector pET23a to form the pET23a-ELP vector. The pET23a-ELP plasmid was transformed into Escherichia coli DH5α strain, and the pET23a-ELP plasmid was extracted after culture and expression. Simultaneously, synthesize 3C protease gene according to the gene sequence (accession number: M12168) of human rhinovirus 3C protease announced on genbank in NCBI, its nucleotide seque...

Embodiment 2

[0034] Embodiment 2: Expression and purification of recombinant ELP-3C protease

[0035] A single colony of the expression host bacterium BL21(DE3) containing the recombinant plasmid pET23a-ELP-3C was inoculated in the LB liquid enriched medium of Amp+, and cultured at 37° C. for 12 hours at 225 rpm to serve as a seed bacterium. Take the seed bacteria and inoculate them in fresh Amp+TB medium at a volume ratio of 1:100, amplify the culture with vigorous shaking at 37°C until the O.D.600 is about 1.0, then adjust the temperature to 18°C ​​for 24 hours. 100ml of the induced bacterial solution was collected by centrifugation at 5000rpm for 5 minutes. The cells were resuspended with 8ml of ice-cold PBS, and then the cells were lysed by sonication at a power of 200W for 15 minutes. The lysed bacteria solution was centrifuged at 12000g at 4°C for 10 minutes to remove insoluble bacterial protein. Add solid NaCl to the centrifuged supernatant and dissolve it. The concentration of th...

Embodiment 3

[0036] Embodiment 3: utilize ELP-3C protease to purify GST protein

[0037] A single colony of pGEX-ELP expression host bacterium BL21(DE3) containing the recombinant plasmid was inoculated in the LB liquid enriched medium of Amp+, and cultured at 37°C and 225rpm for 12 hours as a seed bacterium. The seed bacteria were inoculated in fresh Amp+LB enriched medium at a volume ratio of 1:100, amplified and cultured with vigorous shaking at 37°C until the O.D.600 was about 1.0, then 0.5mMIPTG was added, and the expression was induced at 37°C for 4 hours. After the bacteria were harvested, the solution was lysed, and the concentration of the solution was 2M by adding solid NaCl to trigger the GST-ELP to agglutinate and centrifuge to precipitate it, and 3C digestion buffer was used for dissolution. Add ELP-3C protease at a ratio of 1:100, and digest at 5°C for 16 hours. After digestion, solid NaCl was added to make the solution concentration 2M, triggering the aggregation of ELP and...

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Abstract

The invention relates to an efficient fusion protease, namely an ELP-3C protease which is obtained through the fusion expression between elastin-like polypeptide (ELP) and human rhinovirus 3C protease. As the ELP is adopted as a purification tag, the fusion protease can be purified without the assistance of a chromatography separation technology, thereby reducing the cost of purification. The ELP-3C protease can be applied to cutting fusion protein; and after enzyme cutting of substrate protein, the ELP-3C protease is triggered to precipitate through simply increasing the concentration of salt ions and is removed through centrifuging, wherein the process is performed without the assistance of the chromatographic separation technology, and the ELP-3C protease can be used for the enzyme cutting continuously for multiple times and keeps the enzyme activity after multiple times of the enzyme cutting, thus the cost of large-scale protein purification can be reduced.

Description

technical field [0001] The invention relates to a recombinant fusion protease and the application of the protease in cutting fusion protein. Background technique [0002] With the development and wide application of large-scale sequencing technology, the Human Genome Project and genome projects of other species including sea urchin, zebrafish, and amphioxus have been completed one after another. The direct result of sequencing the genomes of these species is the generation of tens of thousands of genes, the elucidation of the function of these genes helps us understand the position of the species in the evolutionary process; the study of the genes that cause diseases helps to understand the pathogenesis , find therapeutic targets, and develop corresponding therapeutic drugs; at the same time, a large number of drug protein genes are still waiting for us to study their physiological activities and targets, so as to provide a basis for the development of new therapeutic drugs....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/62C12N15/70C12P21/06
CPCC07K2319/50C07K2319/00C07K14/78C12N9/50C07K1/14C12N15/62C12N9/52
Inventor 徐安龙蓝东明黄光瑞王磊陈尚武
Owner SUN YAT SEN UNIV
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