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Design And Purification Of Proteins

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Introduction for beginners to protein design and purification.

Text of Design And Purification Of Proteins

  • 1.DESIGN AND PURIFICATIONOF PROTEINS Biotechnology project, 18/05/09 Marielle Brockhoff, Aurore Lacas , Raphael LieberherrSebastian Olnyi, Morgane Perdomini, Zrinka Raguz,

2. PROTEIN FUNCTIONS Transport (O2)Recognition (antibodies)Structure/Architecture Catalysis (enzymes) Communication (hormon) 3. INSULIN PRODUCTIONIslet of Langerhans ORGANORGANISMTISSUEFUNCTIONS INFORMATIONDNA CELL (and NUCLEUS) 4. GENETIC INFORMATION OF INSULIN DNA Book CHROMOSOME 11 ChapterInsulin Sentence GENE CODON Word 469 lettersC G A T 5. FROM DNA TO INSULIN Codon - DNA - C GA T - Insulin - Gl Gl CyGlyIle Val uns Protein = succession of amino acids Posttranslational modifications Insulin correctly folded functional 6. PROTEIN STRUCTUREPrimary structure Secondary structureQuaternary structureTertiary structure 7. INSULIN STRUCTURE 469 letters 156 amino acids 51 amino acids. two chains linked by disulfide bonds 8. INSULIN FUNCTION Transport of glucoserequires insulin Type 1 diabetes Type 2 diabetes 9. PROTEIN DESIGN Making entirely new or modifying proteins for example as drugs 10. PROTEIN FACTORIES: FROM BACTERIA TO BANANA 11. DIFFERENT ADVANTAGESBacteria: Yeast:Insect cellsMoss cellsMammalian E.coliS.cerevisae cells Costs Cheap Cheap MoreCheap More expensive expensive Setting it up Easy setRelativly MoreMoreMore upeasy set up complicated complicated complicated Large scale Easy to Easy to Easy to scale Easy to scale Difficult productionscale upscale upupup Human-likenoTo a smallVery similarVery similarVery similar modificationextend in proteins MultipleNoNoYes Yes No protein production 12. DIFFERENT MODIFICATION TECHNIQUES Bacteria: viral transformation, artifical competence (chemicals, electroporation) Plants: Agrobacterium, particle bombardment, electroporation, viral transformation Humans, Animals: Chemistry, heat shock, electroporation, viral transformation 13. RECOMBINANT DNA TECHNOLOGY IN THE SYNTHESIS OF HUMAN INSULIN Since 1921: Treatement with insulin derived from animals Bovine & porcine insulin slightly different from human insulin Sometimes inflammation at injection sites Fear: long term complications Solution: Inserting insulin gene into E.coli to produce identical human insulin using Recombinant DNA Technology 14. MANUFACTURING SYNTHETIC HUMAN INSULIN Synthesis of the DNA containing the nucleotide sequences of the A and B polypeptide chains of insulin 15. MANUFACTURING SYNTHETIC HUMAN INSULIN PlasmidPlasmid + restriction enzyme Insertion of the insulin gene into plasmid (circular DNA) Restriction enzymes cut plasmidic DNA DNA ligase agglutinates the insulin gene and the plasmidic DNAPlasmid + insulin gene 16. MANUFACTURING SYNTHETIC HUMAN INSULIN Introduction of recombinant plasmidsinto bacteria: E. coli E.coli = factory for insulin production Using E. coli mutants to avoid insulindegradation Bacterium reproduces the insulingene replicates along with plasmidE. Coli 17. MANUFACTURING SYNTHETIC HUMANINSULIN Formed protein partly of a byproduct the A or B chain of insulin Extraction and purification of A and B chainsbyproduct byproduct Insulin A-chain Insulin B-chain 18. MANUFACTURING SYNTHETIC HUMAN INSULIN Connection of A- and B-chain Reaction: Forming disulfide cross bridges Result: Pure synthetic human insulin 19. INSULIN PRODUCTION TODAY Yeast cells as growth medium Secretion of almost complete human insulin Minimization of complex and purificationprocedures Yeast Insulin 20. PROTEIN PURIFICATION Definition Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture of proteins 21. THE APPLICATIONS OF PURIFIED PROTEINS 22. DEGREE OF PURITYDepends on the application of the protein!!! Industrialapplications: not so strict Food and pharmaceuticals high level required, >99.99% Degree is set by the FDA (Food and Drug Administration) 23. PROPERTIES OF PROTEINS USED FOR THE PURIFICATION Differences in proprieties allow a separation of different proteins Properties come from Amino acids composition Amino adic chain length Structure/shape of the protein(folding of the amino acid chain) 24. Size PROPERTIES OF PROTEINS USED FOR Charge Solubility THE PURIFICATION Hydrophobicity Specific BindingI. Sizeproprieties 25. Size PROPERTIES OF PROTEINS USED FOR Charge Solubility THE PURIFICATION Hydrophobicity Specific BindingI. Sizeproprieties I. s II. Charge + +- +--- - ++ ++- -+- ++ + - + + -+- - -- + +- - +o - 26. Size PROPERTIES OF PROTEINS USED FOR Charge I. S THE PURIFICATIONSolubility Hydrophobicity II. . Specific Binding III. Solubility: pH, T, [Salt]proprieties - +--++ - +-+ + Salt - +-+ - + 27. I. SSize PROPERTIES OF PROTEINS USED FOR Charge II. . PURIFICATION THE Solubility Hydrophobicity III. .Specific Binding IV. Hydrophobicityproprieties 28. I. SSize PROPERTIES OF PROTEINS USED FOR Charge II. . PURIFICATION THE Solubility Hydrophobicity III. .Specific Binding IV. HydrophobicityproprietiesI. S II.. III. . IV..V. Specific binding proprieties 29. PROTEIN PURIFICATION Protein Location Index intracellular: sonication - Filtration extracellular - Gel Filtration Purification: concentrate - Ion Exchange proteins, seperatechromatography proteins- Affinity Filtration andChromatography chromatography 30. ULTRA FILTRATION Use: concentration, desalting of proteins, change buffer Membran: Pore size =10-5 -10-2mm Dialysis 31. CHROMATOGRAPHY Purification using specifique protein properties, as: size, charge, hydrophobicity or biorecognition Stationary phase: inert material, or coated material Mobile phase: buffer 32. GEL FILTRATION Mild conditions (according to protein) With any buffer Isocratic Porous matrix in the spherical beads Small proteins diffuse into pores, stay longer 33. ION EXCHANGE CHROMATOGRAPHY IEX Net surface charge According to pH and the number and exposure of amino acids Charge = 0 at pI pH > pI protein pH < pI protein + 34. STEPS IN IEX Matrix with bound groups that are charged Equilibration: adjust pH in order that protein of interest binds to column Elution by changing the ionic strength or the pH Proteins with highest charge elute latest 35. AFFINITY CHROMATOGRAPHY One step Specific binding between protein and ligand (eg substrate, substrate analogue, inhibitor, cofac tor) His tag binds to metal ions 36. POLY HIS TAG Commonly used for recombinant proteins Ni2+ binds (His)6 Eluting with imidazole 37. INSULIN PURIFICATION Extraction (separation of Bacteria/Yeasts) Purification (separation of other proteins) : Cation exchange chromatographyOD measurement Precipitation with Zinc 38. INSULIN EXTRACTION Secretion of insulin in medium: add sequence to insulin gene Clarification of culture medium: isopropanol added to medium, centrifugation and filtration CENTRIFUGATIONBacteriaMedium withinsulin Medium get rid of Bacteria/Yeasts 39. INSULIN PURIFICATION Ex:Cation exchange Chromatography, SP Sepharose Fast Flow Resin CH2SO3- Total ionic capacity: 180-250mol/ml gel Recommended flow rate: 100-300 cm/h Particle size range: 45-165 m Working pH range: 4-13 Maximum temperature: 30C 40. CATION EXCHANGE CHROMATOGRAPHY Resin Regeneration: 0.5N NaOH => resin is clean Equilibration: 20mM sodium citrate buffer at pH 4.0 => fixationNa+ Mix with insulin diluted with 20mM citrate buffer at pH 4.0 =>positively charged Loading of column and flow rate of 200cm/h => fixation ofinsulin X CH2REGENERATIONNa+ + CH2ADD MIX CH2SO3-SO3-SO3- Y EQUILIBRATION Na+ insulin + +resin 41. CATION EXCHANGE CHROMATOGRAPHY Washing: 20mM citrate buffer => elimination of molecules notfixed Elution: 100mM tris HCl, pH 7.5 buffer, flow rate of 100cm/h=> replacement of insulin by H++ CH2+ELUTION+H CH2CH2SO3-SO3-SO3- + Low HCl concentration + +H Fraction with buffer and no insulin Fraction with insulin 42. DETERMINATION OF FRACTIONS CONTAINING INSULINE OD 280nmAromatic amino acid absorb at 280nm => detection of protein presence in solution A= lC 280nm=0.55 x 104 M-1cm-1 Phenylalanin TryptophanTyrosin 43. PRECIPITATION WITH ZINC Add ZnCl2 to purified insulin and adjust pH to 6 => precipitation Refrigerator (8 C) for at least 6h Centrifugation 5000rpm Drying of pellet => dry insulin Yield for ion exchange chromatography and precipitation: around 75% 44. CONCLUSION Productionof proteins is a big market Example: Lilly Insulin production since 1923 Nessecity of good design and purificationprotocol 45. THANK YOU FOR YOUR ATTENTIONQUESTIONS?