Check patentability & draft patents in minutes with Patsnap Eureka AI!

Method for improving yeast cell surface displaying functional capacity of Infliximab Fab fragments through molecular chaperones

A surface display and molecular chaperone technology, applied in the direction of microorganism-based methods, DNA/RNA fragments, chemical instruments and methods, etc., can solve the problems of insufficient effective assembly and folding, weak yeast display efficiency, etc., to improve functional display , Improving the effect of antigen binding ability

Active Publication Date: 2019-08-30
HUBEI UNIV
View PDF1 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, Sivelle et al. used two GAL1 promoters to simultaneously express the VH-CH1 domain and VL-CL domain of Infliximab, and displayed the complete Fab fragment on the surface of yeast cells after folding and assembling in the endoplasmic reticulum, but found that Infliximab's Functional Fab yeast display efficiency is relatively weak [Sivelle C., Sierocki R., Ferreira-Pinto K., et al., "Fab is the most efficient format to express functional antibodies by yeast surface display", mAbs, 2018, 10( 5):720-9], which may be related to the insufficient efficient assembly and folding of the Fab's VH-CH1 domain and VL-CL domain in the endoplasmic reticulum

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 improving yeast cell surface displaying functional capacity of Infliximab Fab fragments through molecular chaperones
  • Method for improving yeast cell surface displaying functional capacity of Infliximab Fab fragments through molecular chaperones

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the construction of genetically engineered bacteria containing plasmid pFab-2

[0033] The gene fragments of molecular chaperones PDI1, KAR2 and LHS1 of Saccharomyces cerevisiae EBY100 were obtained by PCR cloning method;

[0034] PCR reaction system: 10×KOD buffer, 5 μl; dNTP (2.5mM), 4 μl; primer F (10 μM), 3 μl; primer R (10 μM), 3 μl; Pfu polymerase, 2 μl; template, 1 μl; add ddH 2 0 to 50 μl. PCR amplification system: 95°C, 5min; 95°C, 30s, 55°C, 30s, 72°C, 1min, 25 cycles; 72°C, 5min; 12°C, 10min;

[0035] The PCR products of the molecular chaperones obtained above were recovered with 0.8% agarose gel, respectively, and then digested with EcoRI and XhoI. The enzyme digestion system was: XhoI, 1 μl; EcoRI, 1 μl; 10×Cutsmart buffer, 5 μl; PCR recovered products, 30μl, add ddH 2 0 to 50 μl. After digestion at 37°C for 5 hours, recover with 0.8% agarose gel;

[0036] Then it was ligated with the pESE vector that had been double-digested with restric...

Embodiment 2

[0043] Embodiment 2: Fab-induced expression of genetically engineered bacteria containing plasmid pFab-2

[0044] The yeast transformant transformed with pFab-2 plasmid in Example 1 was inoculated in YNB-CAA-Glucose liquid medium, and cultured overnight at 30°C until OD 600 3.0-4.0, change YNB-CAA-Galactose medium to induce, initial OD 600 0.8, induced at 18°C ​​for 48h.

Embodiment 3

[0045] Example 3: Fab yeast cell surface display efficiency and activity detection of genetically engineered bacteria containing plasmid pFab-2

[0046] For the yeast cells induced in Example 2, two samples were taken from each sample, one was used for the detection of Fab display efficiency, and the other was used for the detection of activity. Take 10 respectively 6 For each yeast cell, wash once with solution A, then wash once with solution B, centrifuge at 4°C, 3000rpm for 2min. One of the cells used for Fab activity detection was incubated with different concentrations of 6×His-TNF-α (expressed and purified in Escherichia coli) at 25°C for 30 min, then washed twice with solution B, and finally washed with 20 μl solution B and 0.15 Resuspend in μl fluorescent antibody Anti-6×His-iFluor 647. Another portion of cells used for Fab display efficiency detection was directly resuspended with 20 μl solution B and 0.15 μl fluorescent antibody Anti-HA-FITC. The resuspended cells...

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 discloses a method for improving yeast cell surface displaying functional capacity of Infliximab Fab fragments through molecular chaperones, and belongs to the technical field of antibody engineering. According to the method disclosed by the invention, a novel III promoter Fab surface displaying carrier is used for co-expressing endoplasmic reticulum molecular chaperone protein Pdi or Kar2 and two domains of VH-CH1 and VL-CL of Infliximab, wherein the two domains of the VH-CH1 and the VL-CL are positioned in endoplasmic reticulum through N-terminal endoplasmic reticulum positioning signal peptide to assemble a natural Fab form fragment, and then through Aga1-Aga2 yeast cell displaying system, the natural Fab form fragment is displayed onto the cell surface. Through co-expressing endoplasmic reticulum molecular chaperone protein Pdi or Kar2, the folding and assembling efficiency of the two domains of the VH-CH1 and the VL-CL of the Infliximab Fab fragment in the endoplasmic reticulum is promoted, and the antigen binding capacity of the yeast cell surface for displaying the Infliximab Fab fragment is obviously increased.

Description

technical field [0001] The invention relates to a method for improving the display of functional Infliximab Fab fragments on the surface of yeast cells by using molecular chaperones, and belongs to the technical field of antibody engineering. Background technique [0002] Monoclonal antibodies are currently one of the most economically valuable biotherapeutic drugs, and are widely used in the treatment of viral infections, immune diseases, and cancer. Yeast cell surface display technology is widely used in the transformation and screening of various forms of antibody libraries because it is a eukaryotic expression host and can be combined with the advantages of high-throughput screening technology of flow cytometry. At present, there are mainly two forms of antibody yeast display fragments, including scFv and Fab. ScFv is the smallest functional structure with antibody activity, which is formed by linking the variable regions of the heavy chain and the light chain of the an...

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): C12N15/81C12N15/66C12N15/62C12R1/865
CPCC12N15/81C12N15/66C07K16/241C07K2317/55C07K2319/35
Inventor 易犁梅萌李俊红汪声晨张桂敏
Owner HUBEI UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com