![Ion exchange chromatography
CM cellulose
cation exchanger
CH2-COO-
CH2-COO-
+
+
+
+
To elute our protein of interest, add increasingly higher amount of salt (increase the ionic
strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged
protein to elute off the column.
CM cellulose
cation exchanger
CH2-COO-
CH2-COO-
+
+
+
+
Na+
Na+
Cl-
Na+2
Cl-
Cl-
Cl-
Na+2
+ Increasing [NaCl]
of the elution
buffer](https://image.slidesharecdn.com/recombinantprotein1-190318072544/85/Recombinant-protein-18-320.jpg)
Recombinant protein
- 1. RECOMBINANT PROTEIN : AN OVERVIEW DR. DEEPA G MURICKEN ASSISTANT PROFESSOR DEPARTMENT OF BIOCHEMISTRY ST. MARY’S COLLEGE THRISSUR
- 3. • Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins. • Modify gene sequences and manufacture useful commercial products. • The formation of recombinant protein is carried out in specialized vehicles known as vectors.. • Many of notable proteins are generated from recombinant DNA, using biomolecular engineering • In many cases, recombinant human proteins have replaced the original animal-derived version used in medicine.
- 4. Human recombinants Human growth hormone (rHGH human insulin (BHI): Humulin from Lilly and Novolin from Novo Nordisk among others largely replaced bovine and porcine insulin for human therapy. •Follicle-stimulating hormone (FSH) as a recombinant gonadotropin preparation replaced Serono's Pergonal which was previously isolated from post-menopausal female urine •Factor VIII: •Erythropoietin •Glucocerebrosidase: •Interferon (IF) Animal recombinants Bovine somatotropin (bST) •Porcine somatotropin (pST) •Bovine Chymosin Viral recombinants •Envelope protein of the hepatitis B virus •HPV Vaccine proteins
- 5. WHY RECOMBINANTS ???? INSTEAD OF OTHER SOURCES • Eg: Insulin can be isolated from different sources like bovine, porcine, hog, sheep etc. They have similar structure but slight different in sequences. • Can cause allergy in some people. • Have to do animal slaughter to isolate insulin. • Very less amount of insulin production • Requires more time. • All these problems are solved by recombinant insulin production
- 6. PRODUCTION OF RECOMBINANT PROTEIN
- 8. PRODUCTION OF RECOMBINANT BOWMAN BIRK INHIBITOR IN E.coli The BBIs of horse gram (Dolichos biflorus) HQSTD E P S E S R V 1 H D M D D D H S v R M D
- 9. • The Bowman-Birk Inhibitors are considered as universal cancer chemopreventive agents •It is still unknown whether the anti-tumoral and radio- protective activities of BBIs are correlated to the inhibitory activity of trypsin/chymotrypsin or other enzymes or to the capability of interacting with other macromolecules. Bowman-Birk Inhibitor Concentrate (BBIC) achieved Investigational New Drug status from FDA in 1992
- 10. 1. NdeI Digestion 2. Ligation pRSET-rHGI 3025bps PT7 MCS HGI-III F1 origin Ampicillin NdeI pRSET-rHGI 3157 bps PT7 MCS HGI-III F1 origin Ampicillin NdeINdeI BamHI HindIII HindIII ATG GAT CAT CAT CAG TCA ACT GAT GAG CCC TCT GAG TCT TCA AAA CCA TGC TGT GAT CAG TGC GCA TGC ACA AAG TCA ATC CCT CCT CAA TGC CGC TGT ACG GAC GTT AGG CTG AAT TCG TGC CAT TCA GCT TGC AGT TCT TGT GTT TGC ACA TTT TCG ATT CCT GCA CAG TGT GTT TGT GTT GAC ATG AAG GAC TTC TGC TAC GCA CCT TGC AAA TCT TCA CAT GAT GAT PROTEIN SEQUENCE M D H H Q S T D E P S E S S K P C C D Q C A C T K S I P P Q C R C T D V R L N S C H S A C S S C V C T F S I P A Q C V C V D M K D F C Y A P C K S S H D D Cloning of HGI-III gene in pRSETC
- 11. Functional expression and purification of recombinant HGI (rHGI) rHGI was expressed in BL21 (DE3)pLys S cells , induced with IPTG 1 M 2 kDa 43.0 29.0 20.0 14.3 6.5 Cell free extracts of pRSET-rHGI.
- 12. Cell lysis: rupture cell wall / plasma membrane, --> release contents (organelles, proteins…) 1. Physical means 2. Sonication 3. Osmotic shock PURIFICATION STRATEGIES
- 13. Main Types of Molecular Interactions Hydrogen Bonds Hydrophobic Interactions (waxy residues: Ileu, Leu, Val, Phe, Trp) Ionic Interactions (charged residues:Asp- Glu- S- Lys+ Arg+ His+) Purification strategy depends on: Solubility Charge Molecular weight Affinity
- 14. Characteristic Procedure Solubility: 1. Salting in 2. Salting out eg: Ammonium sulphate precipitation Ionic charge: 1. Ion exchange chromatography 2. Electrophoresis 3. Isoelectric focusing Polarity: 1. Adsorption chromatography 2. Paper chromatography 3. Reverse-phase chromatography 4. Hydrophobic interaction chromatography Molecular size: 1. Dialysis 2. Gel electrophoresis 3. Gel filtration chromatography 4. Ultracentrifugation Binding specificity: 1. Affinity chromatography
- 15. 1 2 0 5 10 15 20 25 30 35 40 0.5 1.0 1.5 2.0 2.5 3.0 Elution vol (mL) A230 200 400 600 800 Trypsininhibitoryactivity(TIU/mL) Trypsin sepharose affinity chromatography profile of rHGI. ( ) TIU/mL, (▲) A 230. Inset: Native PAGE (10 % T, 2.7 % C) of rHGI at pH 8.3. The gels were stained for A: protein; B: trypsin and C: chymotrypsin by APNE zymography SDS-PAGE (15%T, 2.7%C) profile of purified HGIs. AFFINITY CHROMATOGRAPHY Different types of affinity chromatography: His Tag, GST fusion protein, Maltose binding protein, specific affinities like antigen- antibody reactions, enzyme – substrate interactions etc. 43 29 20 14 6,5 kDa
- 16. Gel Filtration Chromatography 0 20 40 60 80 100 120 140 160 180 200 0.5 1.0 1.5 2.0 2.5 3.0 vol ml 230nm 100 120 140 160 180 200 220 240 260 280 300 TIU/ml An elution profile • Gel-filtration of the protein mixture. 5 ml of protein mixture was loaded onto a Sephadex G-50 column equilibrated with buffer (50 mM Tris HCl, pH 7.5). The column was eluted with buffer at ~1 ml/min, collecting 2.5 ml fractions. The absorbance of each fraction was measured at 230 nm and 280 nm.
- 17. Ion exchange chromatography • Ion exchange resins contain charged groups. • If these groups are acidic in nature they interact with positively charged proteins and are called cation exchangers • If these groups are basic in nature, they interact with negatively charged molecules and are called anion exchangers. DEAE cellulose anion exchanger CH2-CH2 -NH+(CH2CH2) - - - - Negatively charged (acidic) protein or enzyme CM cellulose cation exchanger CH2-COO- CH2-COO- + + + Positively charged (basic) protein or enzyme CH2-CH2 -NH+(CH2CH2)
- 18. Ion exchange chromatography CM cellulose cation exchanger CH2-COO- CH2-COO- + + + + To elute our protein of interest, add increasingly higher amount of salt (increase the ionic strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged protein to elute off the column. CM cellulose cation exchanger CH2-COO- CH2-COO- + + + + Na+ Na+ Cl- Na+2 Cl- Cl- Cl- Na+2 + Increasing [NaCl] of the elution buffer
- 19. Centrifugation Separate proteins by size or density Differential centrifugation - separates large from small particles Isopycnic (sucrose-density) centrifugation - separates particles of different densities Purification of viral coat protein
- 21. Final purification methods • Initial methods included fractionation methods like salt precipitation, precipitation using organic solvents, membrane filtration etc. • Secondary fractionation included ion exchange chromatography, gel filtration, adsorption, affinity methods etc. • The protein intended for drugs and similar uses has to be extremely pure and should be free from even traces of other proteins from the source. • Polishing steps has to be performed in final round like HPLC gel filtration, reverse phase HPLC, ion exchange HPLC etc.
- 22. 9.3849.293 A230 B A Size-exclusion chromatography of seed HGI-III and rHGI. The purified proteins were dissolved in 0.1M Tris-HCl, pH 7.5 and loaded on to a BIOSEP-SEC-S 3000 column HPLC Profile of Size-exclusion BIOSEP- SEC-S 3000 column
- 23. 1. Functional Studies Enzymatic Assays Protein-protein interactions Protein Ligand Interactions 2. Structural Studies • Mass Spectrometry • NMR • Crystallization • CD spectroscopy • Sequence analysis 3. Can perform desired changes for a protein either to study its structure or its function. 4. Synthesis of drugs 5. Synthesis of hormones 6. Production of vaccines 7. Production of carrier molecules 8. Monoclonal antibody production 9. Therapeutic Proteins – Preclinical Studies 10. Production of biosimilar Applications