humanization phagemid construction large-scale. humanization light chainheavy chain a4.6.1 (murine...
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![Page 1: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/1.jpg)
Humanization
Phagemid Construction
Large-scale
![Page 2: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/2.jpg)
Humanization
Light chain Heavy chain
A4.6.1
(murine anti-VEGF antibody)
Y0317
(anti-VEGF antibody Fab fragment)
humanized
![Page 3: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/3.jpg)
Phagemid construction
• phoA alkaline phosphatase promoter
• stII periplasm secretion signal
• geneIII coat proteins of M13 bacteriophage
Light chain
Heavy chain
![Page 4: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/4.jpg)
Periplasm
12~15 nm
Gram negative bacteria cell wall
• Contains only 4% of the total cell protein
• Easy purification by osmotic shock
• Oxidizing environment facilitates the formation
of disulfide bond
periplasm
![Page 5: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/5.jpg)
E. coli strain engineering
• Overexpress chaperone Protein folding– GroEL+GroES– KJE complex (DnaK+DnaJ & GrpE)
• Overexpress disulfide bond enzyme Forming disulfide bond– DsbA
• Lower protease Inhibit protein degradation
![Page 6: Humanization Phagemid Construction Large-scale. Humanization Light chainHeavy chain A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)](https://reader036.vdocuments.mx/reader036/viewer/2022081419/5697c01e1a28abf838cd0ccc/html5/thumbnails/6.jpg)
Alkaline phosphatase promoter
Growth time
Cell number
Fast growth
High phosphate
Low phosphate
InductionHigh phosphate medium
Low expression (cell growth)
NO Need Inducer!!
Low phosphate medium
High expression (induction)