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Expression Vector Expression of cloned genes produces large quantities of protein

Expression Vector Expression of cloned genes produces large quantities of protein. Components of expression vector replication origin polylingker (MRS or MCS) Selective marker promoter operator ribosome binding site gene encoding repressor.

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Expression Vector Expression of cloned genes produces large quantities of protein

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  1. Expression Vector Expression of cloned genes produces large quantities of protein • Components of expression vector • replication origin • polylingker (MRS or MCS) • Selective marker • promoter • operator • ribosome binding site • gene encoding repressor

  2. 그림 19.7 외부 DNA 절편은 제한효소를 이용하여 플라스미드 내로 삽입될 수 있다.

  3. pET vector f1 origin Xho I (7218) Hin dIII (6887) kan Nco I (6676) preCGT Hin dIII (5873) Nde I (5851) pET26-preCGT 7379 bp Nde I (5071) lacI

  4. pET26

  5. pET26

  6. Regulation of Protein Expression in pET System • Double induction by IPTG • T7 RNA polymerase (98 kDa) • target gene (only in T7lac vectors) • Compatible with a wide range of expression hosts • requires DE3 lysogen

  7. Lac operon의 조절 그림 11.1B

  8. Procedure to purify proteins fused with an affinity tag • Fusion of GST gene to target protein gene (C- or N-terminal) • Expression in recombinant strain • Cell disruption to prepare cell extract • Binding of the target proteins to resins via the affinity interaction between affinity tag(GST) and ligand (glutathione)

  9. Electrophoresis gel stained with a protein-specific dye (e.g. coomasie blue) SDS-polyacrylamide gel Purification of RNA polymerize from E. coli Cross-linked polymer polyacrylamide SDS CH3(CH2)11SO4-Na+ acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio.

  10. Expression of K6UbGLP-1 in recombinant E. coli

  11. Protein Purification viaIon Exchange Chromatography

  12. Protein...... Specific Binding Site Charged group - Asp, Glu, Lys, Arg, His Hydrophobic patch - Phe, Trp, Ile, Leu, Val etc Metal chelating group - His, Trp, Cys Size, Shape

  13. Use of chromatography • Production of biopharmaceuticals Pilot and large-scale production of Biopharmaceuticals GE Healthcare Bio-Sciences supplies proven integrated solutions for process chromatography Photograph courtesy of Pharmadule AB

  14. What happens in chromatography? • Molecules to be separated diffuse into the beads • They bind under one set of conditions and are released under (usually) other conditions • Different molecules interact differently Gel Column Liquid-filled gel bead

  15. Gel filtration Size HIC (hydrophobic interaction) Hydrophobicity Ion exchange Charge Affinity Biorecognition Reversed phase Hydrophobicity Separation principles in chromatographic purification 05/nov/02

  16. What is ion exchange chromatography? Ion exchange chromatography is a form of LC that separates molecules on the basis of their charge Useful at all stages of purification and at all scales Controllable High selectivity, high capacity Concentrating, high recovery

  17. Separation by charge • Interaction between opposite charges • Charged groups on the proteins interact with charged groups on the ion exchanger. • Different proteins have different charges and interact differently. • Anion or cation exchange • When the protein is negatively charged, it is an anion - anion exchange • When it is positively charged, it is a cation - cation exchange

  18. Basis for selectivity • Some of the charged regions which will influence ion exchange • Different proteins have different charges and different patterns of surface charge

  19. Effect of pH on charge COOH + R NH3 Hydrogen gained High pH Negative charge - COO + R NH3 Low pH Positive charge Hydrogen lost - COO R NH2

  20. COOH + R NH3 - COO R NH2 - COO + R NH3 Titration curves + acid isoelectric point alkaline excess positive charge balanced positive and negative charge excess negative charge Overall charge on protein 10 3 pH - The overall charge on a protein depends on pH

  21. + Charge on protein - Controlling selectivity by pH Anion exchanger 10 3 pH Cation exchanger

  22. Ion-Exchange Chromatography Example: Cation-exchange chromatography

  23. Purification of Taq DNA polymerase expressed in recombinant E. coli Lane M: Marker proteins Lane 1: E. coli cells before induction Lane 2: E. coli cells after induction with IPTG Lane 3: Soluble fraction after cell disruption Lane 4: Soluble fraction after heat treatment Lane 5: Anion exchange chromatography Lane 6: Cation exchange chromatography

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