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