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Expression Vector Expression of cloned genes produces large quantities of protein Components of expression vector 1. replication origin 2. polylingker.

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Presentation on theme: "Expression Vector Expression of cloned genes produces large quantities of protein Components of expression vector 1. replication origin 2. polylingker."— Presentation transcript:

1 Expression Vector Expression of cloned genes produces large quantities of protein Components of expression vector 1. replication origin 2. polylingker (MRS or MCS) 3. Selective marker 4. promoter 5. operator 6. ribosome binding site 7. gene encoding repressor

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

3 pET26-preCGT 7379 bp kan f1 origin lacI preCGT NcoI (6676) XhoI (7218) HindIII (5873) HindIII (6887) NdeI (5071) NdeI (5851) pET vector

4 pET26

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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 그림 11.1B Lac operon의 조절

8 Procedure to purify proteins fused with an affinity tag 1. Fusion of GST gene to target protein gene (C- or N-terminal) 2. Expression in recombinant strain 3. Cell disruption to prepare cell extract 4. Binding of the target proteins to resins via the affinity interaction between affinity tag(GST) and ligand (glutathione)

9 Electrophoresis Cross-linked polymer polyacrylamide acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. SDS-polyacrylamide gel SDS CH 3 (CH 2 ) 11 SO 4 - Na + Purification of RNA polymerize from E. coli gel stained with a protein-specific dye (e.g. coomasie blue)

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11 Expression of K6UbGLP-1 in recombinant E. coli

12 Protein Purification via Ion Exchange Chromatography

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

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

15 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 Liquid-filled gel bead Column Gel

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

17 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

18 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 Separation by charge

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

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23 NH 3 R COOH + NH 3 R COO + - R NH 2 COO - Low pH Positive charge High pH Negative charge Hydrogen gained Hydrogen lost Effect of pH on charge

24 Overall charge on protein - + NH 3 R COOH + NH 3 R COO + - RNH 2 COO - acid isoelectric point alkaline excess positive charge balanced positive and negative charge excess negative charge The overall charge on a protein depends on pH pH 3 10 Titration curves

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

26 Ion-Exchange Chromatography Example: Cation-exchange chromatography

27 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 Purification of Taq DNA polymerase expressed in recombinant E. coli


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