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Cell culture 04

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  • 1. 4rd Lecture Advanced Aminal Cell Culture 2013 2nd Semester Department of Animal Science Chungbuk National University
  • 2. Syllabus Date Topics September 5, 2013 Introduction : What is Cell Culture? September 12, 2013 Cell Culture As Model System For Research September 26, 2013 Cell Culture For Antibody / Protein Production October 17, 2013 Protein Production/Purification October 31, 2013 Stem Cell I November 14, 2013 Stem Cell II November 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS December 12, 2013 Final Exam
  • 3. Date September 26, 2013 Cell Culture For Antibody / Protein Production 조유미, Madhusumida October 17, 2013 Protein Production/Purification 이미진, 정용호 이영, 윤준호 October 31, 2013 November 14, 2013 Stem Cell I Jia Jia Lin, 염동현 November 28, 2013 Stem Cell II Zhao MingHui,권정우 December 5, 2013 Transgenic Animals Lin Zili, 이상배 December 12, 2013 Genome Engineering/NGS 조유진, 김상욱
  • 4. Protein Purification • Recombinant protein expressed in mammalian cells : 20,000 extra proteins • Desired protein should be purified from other proteins • Based on characteristics of proteins (Size, Surface charge, Affinity…) • Unlike DNA/RNA, Every protein behave differently.. - Purification method should be developed by every single protein.
  • 5. Protein Purification Principles - Ion-exchnage Different protein has different ionic properites (Positive or Negative) - Gel Filtration Seperation based on Protein Size - Affinity Some protein bind specific ligands - UltraFiltration Concentration of Protein / Desalting
  • 6. Ion-exchange chromatography - Purification based on protein surface charge
  • 7. - Anion Exchange Chromatography Resin has (+) charge on it Negative (anion) charged protein bound on the resin By increase of anion (Cl-) concetration, protein eluted from the resin Q-Sepharose : Strong Anion Exchanger DEAE-Sephasrose : Moderately strong anion exchanger
  • 8. - Cation Exchange Chromatography Resin has (-) charge on it Positive (cation charged protein bound on the resin By increase of cation(For example, Na+) concentration, protein eluted from the resin S-Sepharose : Strong cation Exchanger CM-Sepharose : Moderately strong cation exchanger
  • 9. Examples of Ion-exchange chromatography resin Mono-Q and Mono-S : Strong anion (Q) and cation (S) exchanger
  • 10. General Procedure of Ion Exchange Chromatography 1. Load Sample Unbound protein goes to flow through column 2. Wash Sample with low salt buffer 3. Increase Salt Concentration by Gradients Lower Salt : Weak binding Higher Salt : Strong binding
  • 11. Pros It can bind very large amount of sample : Ideal for initial capturing of desired protein Good for the sample concentration If it is optimized, it can increase purification yield significantly General Purpose chromatography (You can use it most of soluble proteins) Cons Sample should be stable at low salt (less than 100mM) buffer Procedure should be optimized (case by case)
  • 12. Affinity Chromatography - Use resin which can bind specifically desired protein - Examples : Protein A sepharose : bind to IgG GSH sepharose : Glutathione transferase Ni-NTA Sepharose : PolyHistidine Chitin bead : Cellulose binding protein - Before genetic manipulation, Affinity chromatography was very limited usages. - But now it is one of major techniques for protein purifications
  • 13. Affinity tag addition using recombinant RNA technology - Most of protein does not have specific affinity bead to bind. - We can add specific tag on protein to facilliate purification Target Protein Gene Affinity tag Gene Affinity Tag Affinity Bead Affinity Tag Target Protein Gene Target Protein Gene
  • 14. PolyHistidine Tag (His-tag) : Can bind to Ni-NTA bead Blue Color From Nickel Glutathione S-transferase Tag (GST-tag) : Can bind to Glutathione Sepharose FLAGTM tag (DYKDDDDK) : Can bind Flag-tag recognize antibody
  • 15. • Pros - Very high level of purification can be achieved - Initial Capturing • - Cons Limited Affinity Resin At least modification of protein is required Cleavage of Tag
  • 16. Size Exclusion Chromatography - Separate protein based on sizes - Small protein enter bead, while big protein migrate fast - Mainly used for the final step of purification (Protein Polishing)
  • 17. • Pros - Can separate protein based on the oligomeric state - Final Polishing of protein • - Cons Seperation depend on the loading volumes Sample should be highly concentrated Limitation of protein amount
  • 18. Multistep purification Affinity chromatography (Initial capturing) Ion exchange chromatography (Intermediate purification) Size Exclusionchromatography (Polishing Step)
  • 19. Measurement of Purity SDS-PAGE Endotoxin detection -Endotoxin : Generally refer ‘bacterial originated lipoplysaccharide’ (LPS) - Induces unwanted immune response - Should be removed for the biologic productions