Mary Shelley's Frankenstein: The Creature's Attempt at Humanization
Humanization
description
Transcript of Humanization
Humanization
Phagemid Construction
Large-scale
Humanization
Light chain Heavy chain
A4.6.1
(murine anti-VEGF antibody)
Y0317
(anti-VEGF antibody Fab fragment)
humanized
Phagemid construction
• phoA alkaline phosphatase promoter
• stII periplasm secretion signal
• geneIII coat proteins of M13 bacteriophage
Light chain
Heavy chain
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
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
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)