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 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?