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Humanization

Humanization. Phagemid Construction. Large-scale. Humanization. Light chain. Heavy chain. humanized. A4.6.1 (murine anti-VEGF antibody). Y0317 (anti-VEGF antibody Fab fragment). Phagemid construction. Heavy chain. Light chain. phoA  alkaline phosphatase promoter.

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Humanization

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  1. Humanization Phagemid Construction Large-scale

  2. Humanization Light chain Heavy chain humanized A4.6.1 (murine anti-VEGF antibody) Y0317 (anti-VEGF antibody Fab fragment)

  3. Phagemid construction Heavy chain Light chain • phoA alkaline phosphatase promoter • stII periplasm secretion signal • geneIII coat proteins of M13 bacteriophage

  4. Periplasm Gram negative bacteria cell wall periplasm • Contains only 4% of the total cell protein • Easy purification by osmotic shock • Oxidizing environment facilitates the formation • of disulfide bond 12~15 nm

  5. 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

  6. Alkaline phosphatase promoter Cell number • High phosphate medium • Low expression (cell growth) Induction Low phosphate Low phosphate medium High expression (induction) Fast growth High phosphate Growth time NO Need Inducer!!

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