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Introduction for beginners to protein design and purification.
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DESIGN AND PURIFICATION OF PROTEINS
Marielle Brockhoff, Aurore Lacas , Raphael Lieberherr Sebastian Olényi, Morgane Perdomini, Zrinka Raguz,
Biotechnology project, 18/05/09
PROTEIN FUNCTIONS
Catalysis (enzymes)
Communication (hormon)
Structure/Architecture
Transport (O2)Recognition (antibodies)
ORGANISM ORGAN TISSUE
CELL (and NUCLEUS)
FUNCTIONS
INFORMATION
DNA
INSULIN PRODUCTIONIslet of Langerhans
CHROMOSOME 11
DNA
Insulin GENE
CODON
≈ Book
≈ Chapter
≈ Sentence
≈ Word
469 letters…..A
T G C A T G
…...G A TC
GENETIC INFORMATION OF INSULIN
- DNA -
- Insulin - Gly
Val
Glu
Gln
Cys
Ile
G A TC
His Th
r Ser
Arg
Asp
Thr
Codon
Insulin correctly folded functional
Protein = succession of amino acids
FROM DNA TO INSULIN
Posttranslational modifications
PROTEIN STRUCTUREPrimary structure
Secondary structure
Tertiary structureQuaternary 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: E.coli
Yeast: S.cerevisae
Insect cells Moss cells Mammalian cells
Costs Cheap Cheap More expensive
Cheap More expensive
Setting it up Easy set up
Relativly easy set up
More complicated
More complicated
More complicated
Large scale production
Easy to scale up
Easy to scale up
Easy to scale up
Easy to scale up
Difficult
Human-like modification in proteins
no To a small extend
Very similar Very similar Very similar
Multiple protein production
No No Yes Yes No
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
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 Plasmid + restriction enzyme
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
Insulin A-chainInsulin B-chain
byproduct
byproduct
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
DefinitionProtein 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)
PROPERTIES OF PROTEINS USED FOR THE PURIFICATION
I. Size
Size
Charge
Solubility
Hydrophobicity
Specific Binding proprieties
PROPERTIES OF PROTEINS USED FOR THE PURIFICATION
I. Size
I. sII. Charge
++
+
++++
+-+++
++
++
--
-
-
--
--
-
-
-
---
+-
+ -o
Size
Charge
Solubility
Hydrophobicity
Specific Binding proprieties
PROPERTIES OF PROTEINS USED FOR THE PURIFICATIONI. SII. .III. Solubility: pH, T, [Salt]
+ Salt
-+
-+
-+
-+
-+
-+
-+
-+
Size
Charge
Solubility
Hydrophobicity
Specific Binding proprieties
I. SII. .III. .IV. Hydrophobicity
PROPERTIES OF PROTEINS USED FOR THE PURIFICATION
Size
Charge
Solubility
Hydrophobicity
Specific Binding proprieties
I. SII. .III. .IV. Hydrophobicity
PROPERTIES OF PROTEINS USED FOR THE PURIFICATION
Size
Charge
Solubility
Hydrophobicity
Specific Binding proprieties
I. SII. .III. .IV. .
V. Specific binding proprieties
PROTEIN PURIFICATION
Protein Locationintracellular: sonicationextracellular
Purification: concentrate proteins, seperate proteinsFiltration and chromatography
Index- Filtration- Gel Filtration- Ion Exchange chromatography- Affinity 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, cofactor)
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
Bacteria
Medium
CENTRIFUGATIONMedium with insulin
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
•CH2
SO3-
X
Y
resin
•CH2
SO3-
Na+
Na+
REGENERATION
EQUILIBRATION
ADD MIX •CH2
SO3-
+
+insulin +
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
SO3-
+
+
•CH2
SO3-
+H
+H
Fraction with insulin
•CH2
SO3-
+
+
Fraction with buffer and no insulin
ELUTION
Low HCl concentration
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 Production of proteins is a big market
Example: Lilly Insulin production since 1923
Nessecity of good design and purification protocol
THANK YOU FOR YOUR ATTENTION
QUESTIONS?
