Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for production of streptavidin labeled phycocyanin fluorescent protein and application

A streptavidin and phycocyanin technology, which is applied in the field of producing streptavidin-labeled phycocyanin fluorescent proteins, can solve the problems of reduced fluorescence and biological activity of fusion proteins, difficulty in experimental synchronization control of expression levels, and plasmids. Loss and other problems, to achieve good practical value and application prospects, reduce production costs, and improve the effect of stability

Inactive Publication Date: 2015-02-25
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is easy to cause the loss of plasmids in actual operation, and it is difficult to control the expression of each gene in an experiment simultaneously, so that the fluorescence and biological activity of the fusion protein are reduced or unstable.

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
  • Method for production of streptavidin labeled phycocyanin fluorescent protein and application
  • Method for production of streptavidin labeled phycocyanin fluorescent protein and application
  • Method for production of streptavidin labeled phycocyanin fluorescent protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the construction of genetic engineering strain

[0032] According to the information in the international gene database, referring to the Chinese invention patent: a preparation method of high-stability phycocyanin fusion fluorescent protein (publication number: CN101838661A), various genes for expression plasmid construction were cloned. The phycocyanin-like apoprotein subunit gene (cpcA), the core streptavidin gene (sa), and the genes encoding phycocyanin cleavage isomerase E and F (cpcE and cpcF) were cloned into the same promoter Next; the heme oxygenase 1 gene (hox1) or its homologous gene, the phycocyanin ferredoxin reductase gene (pcyA) or its homologous gene are placed under another promoter to construct the expression plasmid pAC, After conventional enzyme digestion detection and sequencing analysis, the plasmid was constructed correctly ( figure 1). The pAC was transformed into the host strain BL21(DE3) to obtain the genetically engineered stra...

Embodiment 2

[0034] Example 2: BAC Shake Flask Culture and Induced Expression of Recombinant Protein

[0035] According to the conventional shaking flask culture conditions, different carbon sources were added, and the BAC was cultured to the pre-logarithmic growth stage. Add inducers with final concentrations of 0.2 and 1 mmol / L respectively, induce expression at 18-37°C, 100-350 rpm for 8 hours, collect cells, and observe the color change of cell pellets. As can be seen from Table 1, adding 0.4% glycerol (or 1% glucose) accounting for the weight of the medium in the medium, and when selecting 28°C, when adding an inducer with a final concentration of 5.5mmol / L lactose, the expression level of the target protein is the highest. it is good. However, SDS-PAGE analysis found that most of the recombinant protein existed in the precipitate in the form of inclusion bodies ( figure 2 ).

[0036] Table 1 Effects of different types and concentrations of inducers on the expression of target pro...

Embodiment 3

[0038] Embodiment 3: Purification and preparation of recombinant protein

[0039] The recombinant protein contains a histidine tag, and the protein is purified using a nickel affinity column. The specific operation steps are: after BAC is induced to express, use denaturing buffer (20-50mM Tris-HCl buffer, 8M (mol / L) urea Urea, pH7.3-8.3, 0.15M (mol / L) NaCl, the balance is PBS phosphate-buffered saline), crushed at room temperature for 15-30 minutes, centrifuged, and the supernatant was filtered through a 0.45 μm filter membrane, and then affinity purified with a nickel column. After SDS-PAGE analysis, the purified recombinant protein was dialyzed against 10 L of 0.1 M phosphate buffer (TAKARA) at 4° C. for 48 hours. The purified recombinant protein had only one band, and the size was consistent with the expectation ( image 3 ).

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

No PUM Login to View More

Abstract

The invention relates to the phycocyanin fluorescent protein field, and particularly relates to a method for production of a streptavidin labeled phycocyanin fluorescent protein and an application. A streptavidin synthetic gene, a phycocyanin subunit synthetic gene, a catalytic enzyme synthetic gene and a chromophore synthetic gene are recombined in a same expression plasmid, and combination expression of multiple genes is realized; through optimization of induction and purification conditions, the fusion protein having stable fluorescent activity is prepared. Combination expression of fluorescent activity fusion phycobiliproteins are completed by adopting the single plasmid, the problems that the stability of multiple plasmids in an engineering strain and non-synchronization of expression of genes are avoided, and the use of antibiotics in the fermentation process is reduced. The method can effectively improve the stability of the fluorescent protein, besides, greatly reduces the production cost of phycocyanin fluorescent labeled preparations, and has good practical value and application prospects in the biomedical field, particularly in the production of fluorescent probes.

Description

technical field [0001] The invention relates to the field of phycocyanin-like fluorescent proteins, in particular to a method and application for producing streptavidin-labeled phycocyanin-like fluorescent proteins. Background technique [0002] As a fluorescent marker, phycobiliprotein has the following advantages: good stability, easy storage, no obvious change in the spectrum at pH 4-11, many chromophores, strong light-absorbing ability, high fluorescence quantum yield, and the fluorescence intensity is 14.5 times that of fluorescein. Fluorescence is located in the orange-red region (550-700nm), less background interference, large Stokes shift (fluorescein is less than 30nm, phycobiliprotein is greater than 80nm), isoelectric point is at pH4.7-5.3, negatively charged in physiological solution, There is very little non-specific adsorption, and there are many active groups such as -SH and -NH2 on the surface of the molecule, and it is easy to cross-link. Therefore, phycobi...

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): C12P21/00C07K1/36C07K1/30C07K1/22
Inventor 焦绪栋秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com