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Design and

purification of
proteins
Biotechnology project,
18/05/09

Marielle Brockhoff, Aurore Lacas , Raphael
Lieberherr Sebastian Olényi, Morgane
Perdomini, Zrinka Raguz,

Protein functions
ØTransport (O2)
ØRecognition (antibodies)
ØStructure/Architectur
e
ØCatalysis (enzymes)
ØCommunication
(hormon)

insulin production

ORGAN

ORGANIS
M

Islet of Langerhans

TISS
UE

FUNCTIONS
INFORMATION
DNA

CELL (and
NUCLEUS)

genetic information of insulin
DNA


Book

CHROMOSOME 11


Chapter

Insulin
GENE


Sentence

CODON

.A
.
… .

C
G
T

G
T
A


Word
469 letters
CG AT

from dna to insulin
- DNA
-

Codon

C GA T

- Insulin
-

Gl
y

Ile

Va
l

Gl
u

Gl
n

Cy
s

Protein = succession of
amino acids

Posttranslational
modifications
As
p

Hi
s

Th
r
r

Th
r

S

e

r

A

Insulin correctly
folded

g

Protein structure
Primary structure

Secondary structure

Tertiary structure

Quaternary structure

Insulin Structure
469 letters 156 amino acids 51 amino
acids.
 two chains linked by disulfide bonds

Insulin function
 Transport

of
glucose requires
insulin
ØType 1 diabetes
ØType 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

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
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
byproduc

byproduc

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)

Properties of proteins
used for the purification
I.

Size

Properties of proteins
used for the purification
I.

Size

I. s
II. Charge

++
- +-- +- ++ +++ - - -++ +
+
+
- +
+

+

o

-

Properties of proteins
I. S
used for the purification
II. .

III. Solubility: pH, T, [Salt]

-+

-+

-+
-+

-+
+
Salt

-+
-+

-+

I. S
Properties of proteins
II. .
used for the purification
III. .
IV. Hydrophobicity

I. S
Properties of proteins
II. .
used for the purification
III. .
IV. Hydrophobicity
I.
II.
III.
IV.

S
.
.
.

V. Specific binding
proprieties

Protein Purification
Protein Location
intracellular:
sonication
extracellular
 Purification:
concentrate proteins,
seperate proteins
Filtration 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

CENTRIFUGATION

Medium

get rid of Bacteria/Yeasts

Medium with
insulin

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

X

REGENERATION

SO3-

Y

resin

EQUILIBRATION

•CH2

Na+

ADD MIX

•CH2

SO3-

+

SO3-

Na+

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

+
•CH2

SO3-

+

Low HCl
concentration

+

ELUTION

SO3-

•CH2

+H

SO3-

+

+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
 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?