Expression Vector Expression of cloned genes produces large quantities of protein

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 Vector

5. pET26 Vector Sequence

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. pET expression system Two-step expression vector system target ▪ E. coli BL21(DE3) - Lac promoter (IPTG inducible) fused to T7 polymerase gene ▪ pET-26 vector - T7 promoter fused to target DNA - Target protein: SlyD with 6-Histidine tag at the C-terminus Target cDNA

8. Lac operon의 조절 그림 11.1B

9. 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)

10. 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.

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

13. Protein Purification viaIon Exchange Chromatography

14. 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

15. 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

16. 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

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

18. Column Chromatography Most common method for separating proteins

19. Protein Separation and Purification Column Chromatography • Crude extract --> --> --> fractionation • Salting out (염석): protein solublility, ammonium sulfate (NH4)2SO4 • Dialysis (투석)

20. Exclusion Chromatography (gel filtration)

21. Size-exclusion chromatography

22. Size-exclusion chromatography Absorbance at 280 is used to identify protein-containing fractions. You can also perform an enzyme specific assay.

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

24. - - + + + + + + + + + - + + + + - - - - + + + - + + + + Ion-Exchange chromatography If pH mobile phase =7.2 Then charge of the proteins: (-) (-) (+) (+) Anion exchange column = + charged

25. + + + Cl- + + + Cl- - + + + Cl- - - + + + Cl- + + + Cl- Na+ - Na+ - Na+ Na+ Na+ - - Na+ Na+ + + Na+ Cl- - Na+ Ion-Exchange chromatography - - + + Increased salt concentration

26. 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

27. 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

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

32. 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

33. 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

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

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

36. 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

37. Affinity Chromatography

38. Affinity chromatography Makes use of specific binding interactions between molecules 1- Incubate crude sample with the immobilized ligand 3- Elute 2- Wash away non bound sample components from solid support

39. Affinity chromatography • Commonly used affinity columns: • Ni2+ binds to poly Histidines (example 6xHis) • Specific antibodies (anti-Flag tag) • glutathione  binds to GST • Protein A or G  binds antibodies

40. Affinity chromatography • Possible elution strategies: • pH • Ion strength • Denature • Competitor ligand or analog

41. NTA has a tetradentate chelating group that occupies four of six sites in the nickel coordination sphere Ni-NTA columns The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to: 1- the strength with which these ions are held to the NTA resin

42. 2- the specificity of the interaction between histidine residues and immobilized nickel ions

43. Protein elution Elution of His tagged proteins can be achieved either by reducing the pH, or by competition with imidazole. • Monomers are generally eluted at approximately pH 5.9 or with imidazole concentrations greater than 100 mM, • Multimers elute at around pH 4.5 or 200 mM imidazole.

44. 2+ 2+ Ni Ni Why imidazole? The imidazole ring is part of the structure of histidine

45. Materials and Methods Ni - affinity chromatography Strain : E. coli BL21(DE3) pRIL pET19-pfu E. coli BL21(DE3) ΔslyDpRILpET19-pfu Washing 10ml (Elution buffer) ↓ 2ndwashing 10ml (Washing buffer) ↓ Sample loading ↓ Washing 10ml (Washing buffer) ↓ Elution (elution buffer 2ml) 3times Histidine Imidazole Ni resin