Design And Purification Of Proteins

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?