Design And Purification Of Proteins

DESIGN AND PURIFICATION OF PROTEINS Biotechnology project, 18/05/09 Marielle Brockhoff, Aurore Lacas , Raphael Lieberherr Sebastian Olényi, Morgane Perdomini, Zrinka Raguz,

PROTEIN FUNCTIONS Transport (O2) Recognition (antibodies) Structure/Architecture Catalysis (enzymes) Communication (hormon)

INSULIN PRODUCTION Islet of Langerhans ORGAN ORGANISM TISSUE FUNCTIONS INFORMATION DNA CELL (and NUCLEUS)

GENETIC INFORMATION OF INSULIN DNA ≈ Book CHROMOSOME 11 ≈ Chapter Insulin ≈ Sentence GENE CODON ≈ Word 469 letters C G A T

FROM DNA TO INSULIN Codon - DNA - C GA T - Insulin - Gl Gl Cy Gly Ile Val u n s Protein = succession of amino acids Posttranslational modifications Insulin correctly folded  functional

PROTEIN STRUCTURE Primary structure Secondary structure Quaternary structure Tertiary structure

INSULIN STRUCTURE  469 letters  156 amino acids 51 amino acids.  two chains linked by disulfide bonds

INSULIN FUNCTION  Transport of glucose requires insulin  Type 1 diabetes  Type 2 diabetes http://www.lillydiabetes.com/content/how-insulin-works.jsp

PROTEIN DESIGN  Making entirely new or modifying proteins for example as drugs

PROTEIN FACTORIES: FROM BACTERIA TO BANANA

DIFFERENT ADVANTAGES Bacteria: Yeast: Insect cells Moss cells Mammalian E.coli S.cerevisae cells Costs Cheap Cheap More Cheap More expensive expensive Setting it up Easy set Relativly More More More up easy set up complicated complicated complicated Large scale Easy to Easy to Easy to scale Easy to scale Difficult production scale up scale up up up Human-like no To a small Very similar Very similar Very similar modification extend in proteins Multiple No No Yes Yes No protein production

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

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

MANUFACTURING SYNTHETIC HUMAN INSULIN  Synthesis of the DNA containing the nucleotide sequences of the A and B polypeptide chains of insulin

MANUFACTURING SYNTHETIC HUMAN INSULIN Plasmid Plasmid + 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 DNA Plasmid + insulin gene

MANUFACTURING SYNTHETIC HUMAN INSULIN  Introduction of recombinant plasmids into bacteria: E. coli  E.coli = factory for insulin production  Using E. coli mutants to avoid insulin degradation  Bacterium reproduces  the insulin gene replicates along with plasmid E. Coli

MANUFACTURING SYNTHETIC HUMAN INSULIN  Formed protein partly of a byproduct the A or B chain of insulin  Extraction and purification of A and B chains byproduct byproduct Insulin A-chain Insulin B-chain

MANUFACTURING SYNTHETIC HUMAN INSULIN  Connection of A- and B-chain  Reaction: Forming disulfide cross bridges  Result: Pure synthetic human insulin

INSULIN PRODUCTION TODAY  Yeast cells as growth medium  Secretion of almost complete human insulin  Minimization of complex and purification procedures Yeast Insulin

PROTEIN PURIFICATION Definition Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture of proteins

THE APPLICATIONS OF PURIFIED PROTEINS

DEGREE OF PURITY Depends on the application of the protein!!!  Industrial applications: not so strict…  Food and pharmaceuticals  high level required, >99.99%  Degree is set by the FDA (Food and Drug Administration)

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)

Size PROPERTIES OF PROTEINS USED FOR Charge Solubility THE PURIFICATION Hydrophobicity Specific Binding I. Size proprieties

Size PROPERTIES OF PROTEINS USED FOR Charge Solubility THE PURIFICATION Hydrophobicity Specific Binding I. Size proprieties I. s II. Charge + + - +-- - - ++ ++- -+- ++ + - + + -+ - - -- + + - - + o -

Size PROPERTIES OF PROTEINS USED FOR Charge I. S THE PURIFICATION Solubility Hydrophobicity II. . Specific Binding III. Solubility: pH, T, [Salt] proprieties - + - - + + - + - + + Salt - + - + - +

I. S Size PROPERTIES OF PROTEINS USED FOR Charge II. . PURIFICATION THE Solubility Hydrophobicity III. . Specific Binding IV. Hydrophobicity proprieties

I. S Size PROPERTIES OF PROTEINS USED FOR Charge II. . PURIFICATION THE Solubility Hydrophobicity III. . Specific Binding IV. Hydrophobicity proprieties I. S II. . III. . IV. . V. Specific binding proprieties

PROTEIN PURIFICATION  Protein Location  Index intracellular: sonication - Filtration extracellular - Gel Filtration  Purification: concentrate - Ion Exchange proteins, seperate chromatography proteins - Affinity Filtration and Chromatography chromatography

ULTRA FILTRATION  Use: concentration, desalting of proteins, change buffer  Membran: Pore size = 10-5 -10-2mm²  Dialysis

CHROMATOGRAPHY  Purification using specifique protein properties, as: size, charge, hydrophobicity or biorecognition  Stationary phase: inert material, or coated material  Mobile phase: buffer

GEL FILTRATION  Mild conditions (according to protein)  With any buffer  Isocratic  Porous matrix in the spherical beads  Small proteins diffuse into pores, stay longer

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 +

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

AFFINITY CHROMATOGRAPHY  One step  Specific binding between protein and ligand (eg substrate, substrate analogue, inhibitor, cofac tor)  His tag binds to metal ions

POLY HIS TAG  Commonly used for recombinant proteins  Ni2+ binds (His)6  Eluting with imidazole

INSULIN PURIFICATION  Extraction (separation of Bacteria/Yeasts)  Purification (separation of other proteins) : Cation exchange chromatography OD measurement  Precipitation with Zinc

INSULIN EXTRACTION  Secretion of insulin in medium: add sequence to insulin gene  Clarification of culture medium: isopropanol added to medium, centrifugation and filtration CENTRIFUGATION Bacteria Medium with insulin Medium  get rid of Bacteria/Yeasts

INSULIN PURIFICATION  Ex:Cation exchange Chromatography, SP Sepharose Fast Flow  Resin –CH2SO3-  Total ionic capacity: 180-250μmol/ml gel  Recommended flow rate: 100-300 cm/h  Particle size range: 45-165 μm  Working pH range: 4-13  Maximum temperature: 30°C

CATION EXCHANGE CHROMATOGRAPHY  Resin Regeneration: 0.5N NaOH => resin is clean  Equilibration: 20mM sodium citrate buffer at pH 4.0 => fixation Na+  Mix with insulin diluted with 20mM citrate buffer at pH 4.0 => positively charged  Loading of column and flow rate of 200cm/h => fixation of insulin X •CH2 REGENERATION Na+ + •CH2 ADD MIX •CH2 SO3- SO3- SO3- Y EQUILIBRATION Na+ insulin + + resin

CATION EXCHANGE CHROMATOGRAPHY  Washing: 20mM citrate buffer => elimination of molecules not fixed  Elution: 100mM tris HCl, pH 7.5 buffer, flow rate of 100cm/h => replacement of insulin by H+ + •CH2 + ELUTION +H •CH2 •CH2 SO3- SO3- SO3- + Low HCl concentration + +H Fraction with buffer and no insulin Fraction with insulin

DETERMINATION OF FRACTIONS CONTAINING INSULINE  OD 280nm Aromatic amino acid absorb at 280nm => detection of protein presence in solution  A= εlC ε280nm=0.55 x 104 M-1cm-1 Phenylalanin Tryptophan Tyrosin

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%

CONCLUSION  Productionof proteins is a big market  Example: Lilly  Insulin production since 1923  Nessecity of good design and purification protocol

THANK YOU FOR YOUR ATTENTION QUESTIONS?

Design And Purification Of Proteins

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