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

Method for effectively expressing NaD1 protein by using yeast expression system

A protein, GS115 technology, applied in the field of genetic engineering, can solve problems to be further studied

Inactive Publication Date: 2021-01-08
GUIZHOU UNIV +1
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have also shown that NaD1 protein can mediate the activity against tumor cell lines through specific and high-affinity interaction with phosphoinositide 4,5-bisphosphate, but its detailed mechanism remains to be further studied

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 effectively expressing NaD1 protein by using yeast expression system
  • Method for effectively expressing NaD1 protein by using yeast expression system
  • Method for effectively expressing NaD1 protein by using yeast expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, construction of expression vector and large-scale extraction of recombinant plasmid

[0030] Wuhan Jinkairui Co., Ltd. used EcoRI and NotI restriction enzymes to clone the NaD1 gene into the pPIC9K vector, and transformed the constructed recombinant plasmid pPIC9K-NaD1 into the E. coli TOP10 strain and spread it on an LB plate (containing kana and ampicillin). resistance), use a sterile pipette tip to pick a single colony containing a successful screen and transfer it to 3 mL of LB liquid medium, and culture it overnight on a shaker at 37°C. Afterwards, 700 μL of bacterial liquid was taken for seed preservation, and the remaining bacterial liquid was inoculated into 300 mL of LB liquid medium (containing ampicillin and kana-resistant), and incubated overnight on a shaking table at 37°C. After the bacteria were collected, a large number of plasmids were extracted. The plasmid identified by electrophoresis after enzyme digestion showed that the purity of th...

Embodiment 2

[0031] Embodiment 2, Competent electrotransformation and positive clone screening PCR identification

[0032] Pick a single colony of GS115 on the BMGY streak plate and put it in 15 mL of BMGY liquid medium, and culture it at 200 rpm at 30°C for 24 hours. Inoculate 100 μL of bacterial liquid into 100 mL of BMGY medium, cultivate at 30°C and 200 rpm until the OD600 value is 0.8 to 1.0, pour the cultured bacterial liquid into a pre-cooled centrifuge tube, centrifuge at 3000 g at 4°C for 5 min, and remove the supernatant. Resuspend the bacteria in ice-sterile water, centrifuge at 3000g at 4°C for 5min, and remove the supernatant. Add 1 / 2 volume of ice-sterile water to the above step, and repeat the previous step. After the cells were resuspended in 1M sorbitol on ice and precipitated, repeat the previous step. Resuspend 50 mL of bacterial pellet with 200 μL of ice-cold 1M sorbitol, aliquot into 80 μL / tube of competent cells and store in an ultra-low temperature refrigerator. T...

Embodiment 3

[0037] Example 3, SDS-PAGE detection of small amount of expression products of positive clones

[0038] The clones with positive colonies identified by PCR were selected and stored in ultra-low temperature refrigerators. And the positive strains were picked with a pipette tip and cultured in 1.5mL BMGY liquid medium at 30°C and 200rpm shaker for 48h. After standing the bacterial liquid for 5-6 hours, remove the supernatant, add 1mL BMMY, and incubate at 30℃200rpm for 24h; the induction condition is 200μL of YP medium containing 5% methanol and incubate at 30℃200rpm for 24h; centrifuge at 11000rpm for 10min, take the supernatant and add 150μL TCA ice bath for 2h; centrifuge at 11000rpm for 10min, remove the supernatant, add 200μL acetone to wash the precipitate, repeat the previous step, and dry in an oven at 60°C for 15-20min; add 20μL 1×loading buffer to resuspend and dissolve the precipitate, then use SDS-PAGE detection. The results showed that all positive clones expressed...

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 discloses a method for effectively expressing NaD1 protein by using a yeast expression system. According to the invention, a recombinant plasmid pPIC9K-NaD1 is constructed by connectinga Nicotianaalata NaD1 gene between EcoR I and Not I of a pPIC9K vector through double enzyme digestion and is transferred into GS115 to obtain a recombinant NaD1 protein expression strain; the straincan effectively, stably and massively express recombinant NaD1 protein; and the total amount of the purified recombinant protein is 1 mg, and the protein purity reaches 85%. A product is detected by Western Blot and SDS-PAGE; the actual molecular weight of the recombinant protein is about 20 kDa, and the theoretical value is about 11 kDa; and it is speculated that glycosylated modification of theprotein possibly occurs in pichia pastoris and a polymer form exists. The NaD1 protein is successfully expressed through the yeast expression system, and an experimental basis is provided for furtherresearching an action mechanism of the NaD1 protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for effectively expressing NaD1 protein by using a yeast expression system. Background technique [0002] At present, the production of pharmaceutical proteins by cell engineering has become one of the three major trends in biotechnology and pharmaceuticals in the world. An important scientific research technology in heterologous protein expression system is Pichia pastoris. The time of using this technology in heterologous protein expression can be traced back from 1987 to the present. Studies have shown that more than 500 species come from various aspects. The protein has been secreted and expressed by Pichia pastoris, and the medicinal protein put into industrialization occupies a very important part. Pichia pastoris not only has the characteristics of easy mass reproduction and culture of prokaryotes, but also has the ability of eukaryotes to...

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): C12N15/81C12N1/19C12N15/29C07K14/415C07K1/36C07K1/34C07K1/22C12R1/84
CPCC12N15/815C07K14/415
Inventor 赵懿琛杨德奕龚伟伟赵德刚
Owner GUIZHOU UNIV
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