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
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
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
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
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
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
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
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
Meistens verstecken Forscher die fremden Gene in einem Bakterium. Es dient als \"Vektor\". Auf dem Vektor fährt die Anleitung für den gewünschten Stoff huckepack mit ins Innere der Pflanzenzelle. Dort lösen sich die fremden Gene von ihrem Vehikel und werden in die Pflanzen-DNA eingebaut. Gentechniker zwingen Pflanzen fremdes Erbgut aber auch auf, indem sie DNA an Goldpartikel heften und die Zellen mit dem vergoldeten Erbgut beschießen.
Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1. Property Technique Size Gel filtration (GF), also called size exclusion Charge Ion exchange chromatography (IEX) Hydrophobicity Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) Biorecognition (ligand specificity) Affinity chromatography (AC)
Mild conditions: cold room to 37degree, at any pH, metal ions or co factors, detergents
Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is collected in a purified, concentrated form.