Fusion protein for cellulose accessibility measurement and application thereof

A fusion protein and cellulose technology, which is applied in the direction of fusion with spectrum/fluorescence detection, measurement devices, enzymes, etc., can solve the problems of large measurement deviation, poor repeatability, time-consuming and laborious measurement process, etc., to improve accuracy and avoid changes Effect

Inactive Publication Date: 2017-02-22
TSINGHUA INNOVATION CENT IN DONGGUAN +1
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing technology for measuring cellulose accessibility has the problems that the measured material needs to be dr

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fusion protein for cellulose accessibility measurement and application thereof
  • Fusion protein for cellulose accessibility measurement and application thereof
  • Fusion protein for cellulose accessibility measurement and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Example 1

[0035] Construction of Escherichia coli recombinant plasmid pET28a-GFP2-CBM

[0036] According to the GFP gene and CBM gene sequence in Genebank, two gene sequences of GFP and CBM fusion protein were designed and synthesized on the pUC57-simple vector. According to the gene sequence of the fusion protein GFP2-CBM and the characteristics of multiple cloning sites on the E. coli expression vector pET28a, the following two primers were designed and synthesized:

[0037] GFP_Fe: 5’-GACgaattcATGCGTAAAGGCGAAGAGCTGTTC-3’

[0038] CBM_Rh:5’- GACaagcttTTACAAGCACTGAGAGTAGTAAGG-3’

[0039] The two ends of GFP_Fe and CBM_Rh are respectively designed with EcoRI and HindIII restriction sites, which are expressed in lowercase fonts. Using GFP_Fe and CBM_Rh primers, using the recombinant plasmid pUC57-simple-GFP2-CBM constructed in our laboratory as a template, the GFP2-CBM gene was amplified by PCR. PCR conditions: pre-denaturation at 95°C for 5 minutes; 30 cycles of denaturation...

Example Embodiment

[0041] Example 2

[0042] Expression of fusion protein GFP2-CBM

[0043] The recombinant plasmid pET28a-GFP2-CBM was transformed into E. coli expressing strain BL21, and the bacterial solution was spread on LB plates containing 50 μg / ml Kanamycin for resistance screening, monoclonal PCR detection was selected, and positive clones were selected.

[0044] Pick the recombinant E. coli expression strain BL21-pET28a-GFP2-CBM, inoculate it in 5 ml LB medium, and cultivate overnight at 37°C on a 200rpm shaker. Then transfer 1 ml of bacterial solution to 100 ml of LB medium, and cultivate to OD at 37°C and 200 rpm shaker 600 It is about 0.6, add IPTG to a final concentration of 1 mM, 30℃, 200rpm shaker for 6 hours. The bacterial cells were collected by centrifugation, ultrasonically broken to obtain the supernatant, purified by Ni-NTA medium column, and dialysis to remove salt. The final protein yield was 100 mg / L of bacterial liquid. After SDS-PAGE electrophoresis detection, the target pr...

Example Embodiment

[0046] Example 3 Recognition of pure cellulose by fusion protein

[0047] The fusion protein GFP-GFP-CBM was obtained according to the steps described in Examples 1 and 2. And use similar steps to obtain the fluorescent protein GFP-GFP without CBM. The two proteins, GFP-GFP-CBM and GFP-GFP, were adsorbed on filter paper and microcrystalline cellulose at 37 ℃ and 200 rpm for 2 h respectively, and the adsorbed filter paper cellulose was observed under a fluorescence microscope (attached) figure 1 ) And microcrystalline cellulose (attached figure 2 ), it can be found that due to the recognition effect of CBM on cellulose, the GFP-GFP-CBM fusion protein can be effectively adsorbed on the cellulose, but the GFP-GFP fusion protein does not have this function, so it is not obvious under the fluorescence microscope The phenomenon of fluorescence.

[0048] Result analysis: The results of this example show that the fusion protein provided by the present invention can obtain visual analysis...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention provides a fusion protein for cellulose accessibility measurement and an application thereof. According to the invention, through fusion of recognition functional components of fungus cellulase to a cellulose substrate with a fluorescent protein, the accessibility of cellulose to the fungus cellulase is accurately measured. The fusion protein provided by the invention has a molecular size similar to the molecular size of a key component, namely exoglucanase, of the fungus cellulase, can specifically recognize the cellulose substrate, can obtain fluorescence signals for visual analysis and quantitative measurement, can be used for measuring the accessibility of a substrate under the water-containing state, and avoids needing the step of drying of the substrate in a traditional method for analyzing a pore structure of a solid substance, thereby being able to obtain a more accurate measurement result.

Description

technical field [0001] The invention relates to the field of biochemical industry, in particular to a fusion protein used for measuring the accessibility of cellulose and its application. Background technique [0002] Due to the increasingly serious shortage of fossil resources and the increasingly serious environmental pollution, the production of bioenergy and industrial chemicals from a wide range of sources and renewable lignocellulose has attracted widespread attention from all over the world. Among them, the biotransformation of lignocellulose with biotechnology as the core has become one of the key technologies in bioenergy development, biomass resource processing and green chemical process, and related research has also become a hot spot and difficulty in current industrial microbial technology. However, plants have formed a complex lignocellulose structure to defend against the attack of animals and microorganisms during the long-term evolution process, and the vari...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K19/00C12N15/70G01N21/64
CPCC12N9/2437C07K2319/60C12N15/62C12N15/70C12Y302/01091G01N21/6428
Inventor 赵雪冰李恬欧先金刘德华
Owner TSINGHUA INNOVATION CENT IN DONGGUAN
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products