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

S100B mutant and application thereof

A mutant and protein technology, applied in the field of protein engineering, can solve the problems of low yield and difficult antibody elution, and achieve the effects of high purity, easy elution and separation, and improved recovery rate

Active Publication Date: 2018-06-19
青岛瑞斯凯尔生物科技有限公司
View PDF4 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For the monoclonal antibody purified by the affinity chromatography column coupled with the S100B antigen existing in the prior art, due to the strong binding force between the S100B antigen affinity column and the antibody, the elution of the antibody is very difficult and the yield is low. The invention provides a mutant of S100B, the protein structure is changed, thereby reducing the affinity with the S100B monoclonal antibody. Taking advantage of this feature, the present invention also provides an affinity chromatography column for the purification of the S100B monoclonal antibody, using sepharose 4b is the carrier, coupled with the S100B mutant, can quickly and efficiently obtain high-purity S100B monoclonal antibody

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
  • S100B mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Construction of S100B Gene Mutation Library

[0015] S100B抗原(购自上海友科生物科技有限公司,人工合成)的氨基酸序列如表SEQID No:1所示,即:MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKVMETLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE;其碱基序列如表SEQ ID No:2所示,即:ATGTCTGAGCTGGAGAAGGCCATGGTGGCCCTCATCGACGTTTTCCACCAATATTCTGGAAGGGAGGGAGACAAGCACAAGCTGAAGAAATCCGAACTCAAGGAGCTCATCAACAATGAGCTTTCCCATTTCTTAGAGGAAATCAAAGAGCAGGAGGTTGTGGACAAAGTCATGGAAACACTGGACAATGAT。

[0016] In order to solve the binding force of the S100B antigen (the amino acid sequence is shown in table SEQ ID No: 1, and the nucleotide sequence is shown in table SEQ ID No: 2), the protein was screened for a large number of mutations by directed evolution technology, and optimized And design a pair of PCR primers:

[0017] The upstream primer is as described in the sequence table SEQ ID No: 3, namely: 5'-GGGAGGGAGACAAGCACAGG-3';

[0018] The downstream primer is as described in SEQ ID No: 4 in the sequence table, namely: 5'-GTTCGGATTTCTTCAGCCTGTG...

Embodiment 2

[0019] Construction and fermentation of embodiment 2 S100B mutant expression strain

[0020] 1. Use the S100B gene as a template, use the above primers, and add 2×TransStart FastPfu PCRSuperMix to amplify the entire plasmid gene. The PCR reaction conditions were: denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, renaturation at 60°C for 30 seconds, extension at 72°C for 5 minutes, after 30 cycles, full extension at 72°C for 10 minutes, and storage at 4°C. After PCR, add DMT enzyme to digest and degrade the non-mutant plasmid template in vitro, and recover the target gene by gel.

[0021] 2. Digest the gel-recovered mutant gene and pET-28a vector with BamHI and XhoI to expose the same ends, connect with T4 ligase at room temperature, transform into DMT competent cells, coat LB+Kan plates, and culture overnight at 37°C . The next day, the recombinant strains were screened.

[0022] 3. Pick a monoclonal colony from the culture plate and inoculate it...

Embodiment 3

[0027] Embodiment 3 S100B mutant purification

[0028] Buffer A: 50mM PBS, 500mM NaCl

[0029] Buffer B: 50mM PBS, 500mM NaCl, 500mM imidazole

[0030] 1. After the induced expression, the bacterial solution was centrifuged at 8000rpm for 5min, resuspended in buffer A, ultrasonicated for 30min, and then centrifuged at 12000rpm for 30min to collect the supernatant.

[0031] 2. Add the supernatant to Ni-NTA for 1 hour, use buffer A and buffer B to prepare the imidazole concentration of 10mM, 20mM, 50mM, 200mM buffer to elute the target protein step by step.

[0032] 3. Start the peristaltic pump with a flow rate of 30rpm, turn on the protein detector at the same time, and use various gradient buffer solutions to reach the baseline of the detector until it is stable.

[0033] 4. Collect protein samples from various gradient elutions, and take a small amount of samples to prepare the samples required for SDS-PAGE.

[0034] 5. Collect the target protein, concentrate and change the...

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 belongs to the technical field of protein engineering and particularly relates to a S100B mutant and application thereof. The DNA sequence of the S100B mutant is SEQ ID No:6. A S100B mutant protein has good affinity with an antibody when used as an antigen, and a prepared antibody has high purity. Simultaneously, the S100B mutant protein is easier to elute compared with a non-mutantS100B protein, and the antibody recovery rate is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and specifically relates to the S100B mutant and its application. Background technique [0002] S100B is a low molecular weight (7-11kDa) acidic calcium-binding protein that is mainly synthesized by astrocytes in humans. S100B is a member of the S100 family and contains two chiral EF domains, each of which can bind Ca 2+ , when combined with calcium ions, the conformation of S100 protein changes, exposing its binding site with the target protein, and then exerts biological effects by interacting with the corresponding target protein. As a calcium sensor protein, S100B protein plays an important role in cell proliferation, differentiation, muscle contraction, gene expression and apoptosis through calcium ion signal transduction pathway. In addition, the abnormal expression of S100B can often lead to the occurrence of some neurological, cerebrovascular, tumor and other diseases, such ...

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): C12N15/12C12N15/11C07K14/47C07K16/18C07K1/22B01D15/20B01D15/38
CPCB01D15/20B01D15/3809C07K14/4728C07K16/18
Inventor 孙谧
Owner 青岛瑞斯凯尔生物科技有限公司
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