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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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...
PUM
Property | Measurement | Unit |
---|---|---|
Molecular weight | aaaaa | aaaaa |
Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2023 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap