Advanced Aminal Cell Culture

2013 2nd Semester

Department of Animal Science

Chungbuk National University

4rd Lecture

Syllabus

Date TopicsSeptember 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 INovember 14, 2013 Stem Cell IINovember 28, 2013 TG/KO Animals December 5, 2013 Genome Engineering/NGS

December 12, 2013 Final Exam

DateSeptember 26, 2013 Cell Culture For

Antibody / Protein Production

조유미 , Madhusumida

October 17, 2013 Protein Production/Purification 이미진 , 정용호

October 31, 2013 이영 , 윤준호November 14, 2013 Stem Cell I Jia Jia Lin, 염동현

November 28, 2013 Stem Cell II Zhao MingHui,권정우December 5, 2013 Transgenic Animals Lin Zili, 이상배December 12, 2013 Genome

Engineering/NGS 조유진 , 김상욱

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.

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

Ion-exchange chromatography

- Purification based on protein surface charge

- Anion Exchange Chromatography

Resin has (+) charge on itNegative (anion) charged protein bound on the resinBy increase of anion (Cl-) concetration, protein eluted from the resin

Q-Sepharose : Strong Anion ExchangerDEAE-Sephasrose : Moderately strong anion exchanger

- Cation Exchange Chromatography

Resin has (-) charge on itPositive (cation charged protein bound on the resinBy increase of cation(For example, Na+) concentration, protein eluted from the resin

S-Sepharose : Strong cation ExchangerCM-Sepharose : Moderately strong cation exchanger

Examples of Ion-exchange chromatography resin

Mono-Q and Mono-S : Strong anion (Q) and cation (S) exchanger

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 bindingHigher Salt : Strong binding

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)

Pros

Cons

Sample should be stable at low salt (less than 100mM) buffer

Procedure should be optimized (case by case)

Affinity Chromatography

- Use resin which can bind specifically desired protein- Examples :

Protein A sepharose : bind to IgG GSH sepharose : Glutathione transferaseNi-NTA Sepharose : PolyHistidineChitin bead : Cellulose binding protein

- Before genetic manipulation, Affinity chromatography was very limited usages.

- But now it is one of major techniques for protein purifications

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

Affinity tag Gene

Target Protein Gene

AffinityTag Target Protein Gene

AffinityTag Target Protein GeneAffinity Bead

PolyHistidine Tag (His-tag) : Can bind to Ni-NTA bead

Glutathione S-transferase Tag (GST-tag) : Can bind to Glutathione Sepharose

FLAGTM tag (DYKDDDDK) : Can bind Flag-tag recognize antibody

Blue Color From Nickel

• 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

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)

• 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

Multistep purification

Affinity chromatography(Initial capturing)

Ion exchange chromatography(Intermediate purification)

Size Exclusionchromatography(Polishing Step)

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