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La Revolucin de

la Biotecnologa

Prof. Juan A. Asenjo

Edward Jenner (1749 1823): cowpox smallpox Vacuna viruela


1850 Luis Pasteur:
Microorganismos: fermentacin no es espontnea
fermentacin
levaduras
Esterilizacin (descubri los microorganismos)
(Enzimas)
azcar
levadura

CO2 + H2O
alcohol

1928: Alejandro Flemming : Penicilina


1939: Florey, Chain purificacin de penicilina y produccin masiva
USA-Pfizer Produccin de cido ctrico
1945: Premio Nobel: Flemming, Florey, Chain

Obtencin de Plasmidios
1.- Se cuenta con bacterias que contienen plasmidios
Bacteria
Plasmidio
Cromosoma

2.- Sacar plasmidio desde bacteria


Poracin

Plasmidios
Produccin & Purificacin de Protenas

Principales pasos en la Clonacin de


un Segmento de DNA Forneo
1.- Obtencin del DNA forneo
Extremos
cohesivos

Extremos
cohesivos

2.- Corte con Enzimas de restriccin del plasmidio


Extremos
cohesivos

Corte
(Enzimas de
Restriccin)
Plasmidio

Plasmidio Cortado
Produccin & Purificacin de Protenas

60s - 70s Ingeniera Gentica


80s INSULINA: Ingeniera gentica de E.coli y S.cerevisiae
Insulina comercial recombinante
Hoy: Eli-Lilly

Novo-Nordisk
90s: tpA
Vacunas: Contra hepattis B (Merck, Chiron)
Sida
1990 Sally y Dolly
Enzimas crioflicas
Terapia celular y gnica

Nueva Biologa Molecular


Protenas Clonadas
Ingeniera Gentica
Enzimas de Restriccin
Plasmidios

Produccin & Purificacin de Protenas

Principales pasos en la Clonacin de


un Segmento de DNA Forneo
1.- Obtencin del DNA forneo
Extremos
cohesivos

Extremos
cohesivos

2.- Corte con Enzimas de restriccin del plasmidio


Extremos
cohesivos

Corte
(Enzimas de
Restriccin)
Plasmidio

Plasmidio Cortado
Produccin & Purificacin de Protenas

4.- Transformacin
4a.- Permeabilizacin de la clula mediante permeasa

Permeasa

4.b.- Introduccin del plasmidio Recombinante en clula anfitriona

Produccin & Purificacin de Protenas

Biotecnologa

Nueva Biologa Molecular


Protenas Clonadas
Ingeniera de Protenas
Ingeniera Metablica (Systems Biology)
Genmica Funcional

Nuevos Productos Teraputicos


Nuevas Vacunas
Nuevas Enzimas Industriales
Cultivo de Tejidos, Terapia Gnica

We havent the money, so weve got to


think
Ernest Lord Rutherford, 1871 - 1937

Systems Biology

Ogni parte ha
inclinazione di
ricongiungersi al
suo tutto
per fuggire dalla
sua imperfezione
Leonardo da Vinci
(Cod.Atl, fol 59 recto)

The part always


has a tendency
to reunite with
its whole in order
to escape from
its imperfection
Leonardo da Vinci
(Cod.Atl, fol 59 recto)

Estructura de las Protenas


Estructura Primaria: secuencia lineal de aa
Estructura Secundaria: algunos aa interactuan
Estructura Terciaria: cadenas de aa interligadas
Estructura Nativa: protena se encuentra activa
Protena denaturada:
No tiene actividad
No posee puentes dislfuro

Produccin & Purificacin de Protenas

Protenas
Cuatro niveles de
estructura:
desde 1 dimensin
a 3 dimensiones
Desde anlisis
estructural
a anlisis funcional

Ingeniera de Proteinas: Prediccin Estructural

Ingeniera de Protenas

Proteasa crioflica antrtica

Ingeniera de Protenas
Proteasas activas a baja temperatura
(Crioflicas, Psicroflicas)
para detergentes
para industria de alimentos
Para aplicaciones mdicas

Ingeniera de Protenas
Estudios de Relacin Estructura-Funcin
Mutagnesis Sitio-Dirigida
Mutagnesis al Azar

Mutagnesis al azar (random)


Evolucin dirigida
Mutagnesis de saturacin
Gene shuffling
(barajar genes)

Proteasa crioflica antrtica

Is there a Rational Method to


Purify Proteins?
from Expert Systems to
Proteomics

J.A. Asenjo
University of Chile

The Combinatorial Characteristic of Choosing the


Sequence of Operations for Protein Purification
First
Stage

Second
Stage

Third
Stage

n th
Stage

1) Ion Exchange
Chromatography
2) Hydrophobic
Interaction
Chromatography

A1

B1

C1

n1

A2

B2

C2

n2

A3

B3

C3

n3

A4

B4

C5

n5

A6

B5

C6

n6

B6

3) Affinity
Chromatography
4) Aqueous TwoPhase Separation
5) Gel Filtration
6) HPLC

The architecture of a knowledge based expert system. (taken from


Asenjo, Herrera and Byrne, 1989)
Knowledge base
Rules

Facts

Working
memory

Inference engine
Inference

Knowledge
acquisition
subsystem
Expert or
Knowledge
engineer

Control

Explanation
subsystem

User
interface

User

Determination of the Resolution Between Two Peaks

V2

RS =

Absorbance

V1

V2-V1
(W1+W2)

SC RS

W1

W2

Time

= SC RS
DF
DF

The model of database components for main protein contaminants in one of the
production streams to be used in the selection of optimal separation operation
PROPERTY
CONCENTRATION
PROTEINS
PRODUCT
CONTAMINANT 1
CONTAMINANT 2
CONTAMINANT 3
CONTAMINANT 4
CONTAMINANT 5

......
CONTAMINANT N

MOLECULAR
WEIGHT

ISOELECTRIC
POINT

HYDROPHOBICITY

CHARGE

pH 4.0 pH 4.5 . . . . pH 9.5 pH 10.0

Concentration, molecular weight, hydrophobicity and charge at different pHs, for the main
proteins (contaminants of the product) in Escherichia coli. Data from Woolston (1994)
Charge4 (Coulomb per molecule x 1E25)
g/litre

Da

pH 4

pH 4,5 pH 5 pH 5,5 pH 6 pH 6,5

pH 7

pH 7,5

pH 8

pH 8,5

qE

qF

qG

qH

qI

qJ

-0.80 -1.41

-1.76

-1.97

-2.15

-2.33

-2.45

-2.67

0.29

-1.17 -2.17

-2.83

-3.24

-3.50

-3.63

-3.68

-3.64

1.83

0.67

0.04

-0.30

-0.49

-0.65

-0.85

-1.90

-1.34

-1.50

1.50

3.29

1.38

-0.03 -0.69

-1.07

-1.34

-1.73

-2.30

-2.85

-2.75

203,000

0.36

4.08

1.83

0.04

-1.17

-1.92

-2.46

-3.07

-3.90

-4.98

-5.65

2.48

69,380

0.36

5.22

3.17

1.02

-0.72

-1.90

-2.60

-3.05

-3.46

-3.90

-4.24

5.29

7.70

48,320

0.48

3.96

3.16

1.12

-0.58

-1.36

-1.34

-1.00

-0.95

-1.59

-2.84

Cont_8

5.57

6.80

93,380

0.93

10.90 5.81

2.78

0.77

-0.81

-2.18

-3.32 -4.12

-4.45

-4.31

Cont_9

5.65

7.53

69,380

0.55

0.26

0.10

-0.03

-0.12

-0.21 -0.28

-0.32

-0.32

Cont_10

6.02

6.05

114,450

0.63

10.40 5.94

3.15

1.51

0.56

-0.05

-0.53

-0.99

-1.43

-1.72

Cont_11

7.57

3.89

198,000

0.06

0.33

0.03

0.05

0.05

0.05

0.05

0.05

-0.69

-0.97

-1.57

Cont_12

8.29

1.48

30,400

5.17

4.22

3.20

2.25

1.46

0.87

0.50

0.30

0.20

0.08

Cont_13

8.83

0.83

94,670

11.70

7.94

5.39

3.73

2.66

1.97

1.50

1.13

0.80

0.51

pI 1

weight Mol wt 2 hydroph 3 q A

Cont_1

4.67

11.29

18,370

0.71

Cont_2

4.72

7.06

85,570

Cont_3

4.85

4.63

Cont_4

4.92

Cont_5

qB

qC

1.94

0.25

0.48

2.35

53,660

0.76

5.58

120,000

5.01

4.83

Cont_6

5.16

Cont_7

Contaminant

* Hydrophobicity

1.09

qD

expressed as the concentration (M) of ammonium sulphate at which the protein eluted.
(Higher values represent lower hydrophobicity).
1 Measured by isoelectric focusing using homogeneous poolyacrylamide gel in Phast System.
2Molecular weight was measured by SDS-PAGE with PhastGel media in Phast System.
3Hydrophobicity was measured by hydrophobic interaction chromatography using a phenyl-superose gel in an
FPLC and a gradient elution from 2.0 M to 0.0 M (NH4)2SO4 in 20 mM Tris buffer.
4Charge was measured by electrophoretic titration curve analysis with PhastGel IEF 3-9 in a Phast System.

DFi

DFi
B

S
C

D
b

DFi

DFi

Representation of the peaks of a chromatogram as triangles, showing how the variation in the value of DF
leads to different concentrations of the contaminant protein in the product. The triangle on the left
corresponds to the product protein and the triangle of the right corresponds to the peak of the protein
being separated (contaminant).

Genomics
Proteomics
Metabolomics
Economics

Genes
Protenas
Metabolismo
Sistemas Multicelulares
Organismo

Metabolmica
Ingeniera Metablica
Systems Biology: qu viene
despus de la Genmica
Uso de Anlisis de Flujos
Metablicos y Tecnologa de
Microarrays de Genes

Metabolomics

RNA

SOD
71-aaRIB 5P
70-aaRIB 5P

72-nuRIB5P

aa
PRO T
RIBU5P

nu
RI B5P

19

20

RIB5P

GAP

22
E4P

FRUC6P

aa

70-aaE 4P

28

30
73-AcCoA

AcCoAcit

70-aaAcCoA

- aa O

70 - a

aa
AC

OA

aa
C

RNA

nu

O2 E

69
77

ADP
O2

SOD

aa
aa

PIR

aa

71-aaPIR

25

10

PRO T

CO2

10

17

AcCoAcit
24

14

76

NH4
CO2 E

ISOCIT
11

13

15

78

NH4 E

AKG

SUC

SOD

70-aaPIR

OAC

16

PRO T

70 -aaPE P

75

OAC

MAL

71 -aa3PG

71-aaPE P

PIR

FUM
ATP

72-nu3P G

AcCoAmit

PRO T

72-nuOAC

RNA

nu

PEP

GA

70-aa3PG

PEP

26

71-aaAcCoA
71

3P G

3PG

Ac CoAci t

SOD

73 -

ACET

LIP

GAP

SOD

AC
LIP

FRUC6P

23

E4P

27

CARB

GL IC

EtO H

74

GLUC6P

31

71 -aaE 4P

PRO T

18

XIL5P

21

SED7P

GLUC

SUCCoA

AK G

PRO T

aa

70-aaAKG

71-aaAKG

SOD

Metabolic Flux Analysis


Metabolic Flux Balance
in SS: S v = b

dX/dt = S v - b
or
Sr =0
Sc rc + Sm rm = 0
C

4
5

1 2 3 4 5

S r=0=

S
r
c
m

-1 -1

-1

-1

1
2
3
4
5

Stoichiometric Matrix
Rate (Flux) vector
Calculated
Measure d

1 2 3
B

-1 -1

1
2 +
3

4 5
B

-1

-1

4
5

15

3.5

15

3.5

12

2.8

12

2.8

2.1

2.1

1.4

1.4

0.7

0.7

0.0

0
0

18

27

36

0.0

Glucose, g/L

45

18

Time, h

27

36

45

Time, h

Strain P+

Strain P-

0.20

0.9

0.15

0.6

0.10

0.3

0.05

0.0

Total RNA, g/L

1.2

1.5

0.25

1.2

0.20

0.9

0.15

0.6

0.10

0.3

0.05

0.00
0

18

27
Time, h

36

45

0.0

0.00
0

18

27
Time, h

36

45

Total RNA, g/L

0.25

Total Protein and Carbohydrates, g/L

1.5
Total Protein and Carbohydrates, g/L

Cells and Ethanol, g/L

Strain P-

Cells, Ethanol and SOD, g/L

Glucose, g/L

Strain P+

P+ GLUC

RNA

SOD

0.042 0.006

nu
0.057

0.006

aa
PRO T
RIBU5P
0.234

RIB5P

GLUC
0.559

4.611

0.325

0.177

GLUC6P

XIL5P

0.208

CARB

3.844

FRUC6P
SED7P

0.177

GAP

4.169

0.148

0
0.0

GAP
2.232

E4P

FRUC6P

6.256

GL IC

3PG

0.029

LIP

0.025

EtO H
0 102

aa

0.004

4.130

SOD

ACET
0 137

PEP
4.267

0.105
0.029

6.122
0.138

PIR

RNA

nu

6.151

PRO T

0.019

SOD

aa

0.028

0.048

aa

PRO T

0.025

0.004

SOD

aa

0.017

0 069

PRO T

PRO T

0.025

aa

SOD

0.004

0.029

PEP

EtO H
0.102

LIP

ACET

RNA

0.063

AcCoAcit

0.057

RIB5P

RNA

P+ GLUC

AC

0.177

SED7P

CARB
0.247 AcCoA
mit
3.844

nu

0.177

0.025

6.256

GL IC

3PG

SOD

0.004

OAC
PEP

0.137

0.166

AcCoAcit

aa

0.022

RNA

0.014

4.267

ACET

1.397

0.102

0.063

aa

4.130

EtO H

PRO T

1.397
0.046

PRO T

ATP
O2

8.988

E 3.564

ADP

FUM
ATP

AC

0.025

aa
0.079 MAL

ADP

1.349

0.048

aa

PRO T

CO2

0.025

0.004

SOD

0.138

CO2 E

0.017

0.069

PRO T

ISOCIT
NH4

OAC

0.724
E

CO2

8.850

NH4

1.470

CO2 E

1.470

1.397

8.850

NH4

1.470

aa

1.470

ISOCIT
1.470

FUM

O2 E 3.564 O2

1.470
SOD

0.247 AcCoAmit

AKG

0.121

PRO T
1.349

1.349

SUC

O2

PIR

NH4 E

0.028

aa

nu

1.397

8.988

0.029

0.019

1.470

0.174

SOD

0.105

6.151

0.724

RNA

nu

6.122

aa
0.079 MAL
LIP

02
0 .0

GAP

E4P

LIP

4.169

0.148

0.029

PRO T

0.208

FRUC6P

GAP

0.174

0.046

PRO T

4.611

GLUC6P

XIL5P

FRUC6P

0.025

0.069

1.470

2.232

SOD

0.017

GLUC
0.559

0.325

0.022

0.014

aa

0.006

aa
PRO T
0.166
RIBU5P
0.234

aa

SOD

0.138

PIR

0.137

SOD

0.042 0.006

nu

4.267

0.025

0.004

6.122
4.130

aa

SUCCoA
1.345

SUC

aa

AKG

PRO T

0.097

0.023

SOD

1.349

SUCCoA
1.345

0.121

aa

0.097

0.023

SOD

RATIO P-/P+ GLUC


RNA
1.11

nu
1.10

1.82(0.96)

aa
PROT
RIBU5P
1.63

RIB5P

GLUC
1.74

1.05

1.82

GLUC6P

XIL5P

1.80

1.60

CARB

0.92

FRUC6P
SED7P

GAP

1.80

0.99

1.84

9
4.4

GAP
0.36

E4P

FRUC6P

LIP

GLYC

1.60

RNA
1.11

nu

1.23

3PG

1.23

aa

1.82(1.16)

1.23

PROT

aa

LIP
1.46

PEP

1.82(1.60)

EtOH

1.00

ACET

4.49

AC

RNA

PYR

aa
aa

1.82(1.60)

1.82(1.46)

PROT

1.60
1.40

AcCoAmit

nu
1.60

1.32

1.82(1.41)

PROT

1.09

PROT

3.34
1.11

1.8

1.23
1.39

3.73

AcCoAcit

aa

1.60

NH4E

OAC

)
.39 aa
2(1

1.61

MAL
1.61

FUM

CO2

1.60

1.33

1.38

ISOCIT
1.60
1 47

NH4
CO2E

RNA
0.042
0.012
0.002

0.006
0.000
0.007

0.006
0.003
0.001

aa

nu

P+ : GLUC, EtOH-Exp., EtOH-Stat.

SOD

PROT

0.325
0.109
0.137

0.234
0.075
0.082

RIB5P

GLUC

0.559
0.185
0.219

RIBU5P

0.057
0.015
0.011

4.611
0.000
0.000

GLUC6P

XIL5P
0.148
0.050
0.065

GAP

FRUC6P
0.000
0.140
0.109

0.177 0.060 0.072


0.029
0.010
0.007

FRUC6P
0.025
0.010
0.002

aa

PROT
EtOH
AC

0.004
0.000
0.004
4.130
-1.826
-0.598

SOD
4.267
0.000
0.000

0.137 1.826 0.598

0.046
0.019
0.007

SOD

0.014 0.000 0.025

0.02
5 0
.000
0.03
1
0.079
0.033
0.008

AcCoAcit
0.063
0.021
0.021

aa

aa

0.174
0.053
0.046

0.022
0.006
0.001

MAL

RNA

0.029
0.010
0.007

6.122
0.043
0.577

PYR

0.000
0.531
0.000

0.019
0.006
0.001

nu

0.138
0.043
0.051

0.048 0.020 0.005

PROT
aa

0.025 0.010 0.002


0.004 0.000 0.004

SOD
aa

0.017 0.000 0.030

1.470
0.000
0.526

0.000
1.668
0.394

SOD

0.028 0.000 0.035

aa

PEP

0.069 0.028 0.009

PROT

AcCoAmit
0.247
0.000
0.000

OAC

PROT

0.105
0.029
0.043

3PG
6.151
-0.478
0.584

0.166 1.836 0.608


0.102 0.047 0.079

LIP

6.256
-0.449
0.626

GLYC

ACET

LIP

4.169
2
0.000
.0 0
1 0
0
0.000
0
0.
02
0.0

GAP

2.232
0.218
-0.780

E4P

CARB

3.844
-0.250
-0.246

0.177 0.060 0.072

SED7P

0.208
0.065
0.027

NH4E

1.470
1.158
0.956

CO2

1.397
1.742
1.001

ISOCIT
AcCoAcit

1.470
0 548

0.724
0.221
0.221
8.850
1.774
2.476

NH4
CO2E

etoH
glu cos e

PEtOH/Gluc
Glucose

Ethanol

Central metabolic
pathway
82 genes

Discrete mathematical models


applied to genetic regulation and
metabolic networks

Gene network
Traditional
technologies
Models
Microarrays

Metabolic network
Metabolic
Flux
Analysis

Phenomena to model
Genetic and metabolic
adaptation of E. coli to 3
different carbon sources
Substrates: Glucose, Glycerol
and Acetate
Glycolysis and TCA
8 possible substrate combinations
8 Phenotypes

Phenomena has been


described using Microarrays
(MA) and Metabolic Flux
Analysis (MFA)

Genes regulando el
metabolismo
Estados
Gen
Signal1

Signal2

Gene

Flujo Metablico de Enzima


Comp B1

Enz

Activo

-1

Inactivo

1/2/3
Enz1

0
-1 / -2 / -3

Seales = Biochemicals / Reguladores


Enz1 /
Signal1

Signal

1 / 2 / 3 Activo
Enz2 /
Signal2

Inactivo

Estudio de dinmica
del modelo
67 nodos
28 genes
21 enzimas
18 reguladores / compuestos
bioqumicos

Reguladores Ficticios
para que modelo
alcance Fenotipos
Algoritmo
Definir combinacin de sustratos
Generar 105 vectores aleatorios
Actualizar en forma paralela
Alcanzar atractor

Atractor = Phenotype
Each atractor of model
corresponds to a phenotype
Atractors are very stable
Similarity between Atractors
with:
Glucose present
Glycerol present and Glucose
absent
Only Acetate present and All
others absent

Cultivo de Tejidos
- tejidos
- clulas (e.g. sanguneas)
- rganos

Clulas para Terapia Celular


Vectores para Terapia Gnica

Terapia Gnica

Alcoholismo
Osteoporosis
Parkinson
Cancer (e. mama - gene BRCA-1)
Artritis
Hemochromatosis
Alzheimer

Vector de Primera Generarin

Vector de Tercera Generacin o gutless

Multiproduct and multihost batch


plant

Centrifuge

Homogenizer

Centrifuge

IB Solubilization
Fermentation
Microfilter

Bead mill

Diafiltration

Microfilter

Ultrafilter
Sterile filtration

Ultrafiltration diafiltrater

Ultrafiltration diafiltrater
Chromatography

Sulfonation

Chromatography

All the hosts

E. coli only

Yeast intracellular and E. coli

E. coli and CHO cells

Refolding

Chromatography

OPTIMAL PROCESS SYNTHESIS FOR THE PRODUCTION OF


MULTIPLE RECOMBINANT PROTEINS
Iribarren, Montagna, Vecchietti, Andrews, Asenjo and Pinto