Introduction for beginners to protein design and purification.

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

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

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

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

5. 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

6. PROTEIN STRUCTURE
Primary structure
Secondary structure
Quaternary structure
Tertiary structure

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

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

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

10. PROTEIN FACTORIES: FROM BACTERIA TO
BANANA

11. 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

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
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

16. 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

17. 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

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 purification
procedures
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 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)

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 Binding
I. Size proprieties

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

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

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

28. 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

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

30. ULTRA FILTRATION
 Use:
concentration, desalting
of proteins, change
buffer
 Membran: Pore size =
10-5 -10-2mm²
 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 chromatography
OD 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
CENTRIFUGATION
Bacteria Medium with
insulin
Medium
 get rid of Bacteria/Yeasts

39. 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

40. 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

41. 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

42. 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

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 purification
protocol

45. THANK YOU FOR YOUR
ATTENTION
QUESTIONS?