Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40...
Transcript of Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40...
![Page 1: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/1.jpg)
Concept : Cell Yield
Glucose, mM
0
50
100
150
200
250
300
0 10 20 30 40
Slope = 7.2 µg/ml per mM
Experimental observation –
Cell mass is proportionalto available substrate
![Page 2: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/2.jpg)
Cell Yield – Formal Definition
dS
dXY s/x =
Cell Yield is:
ConsumedSubstrate
MassCellinChangeY s/x =
![Page 3: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/3.jpg)
Cell Growth in Batch Culture
![Page 4: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/4.jpg)
Typical Growth RatesGrowth
RateDoubling
timeµ [h-1] [h]
E. coli 2 0.35Yeast 0.3 2.3Hybridoma 0.05 13.9Insect Cells 0.06 11.6
Organism
![Page 5: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/5.jpg)
Nature of Specific Growth
SK
S
s
m
+
µ=µ
S, g/ L
µ , 1
/h
0
0.1
0.2
0.3
0.4
0.5
0 5 10 15 20 25
Monod Kinetics
Monod Kinetics. Dependence of Growth Rate onLimiting Substrate. Specific growth rate reaches amaximum value of 0.5 h-1. Value of KS here is 0.5 gL-1. Note that when S = 0.5 g L-1, µ is half of itsmaximum.
How does one experimentallydetermine cell parameters?
Population Growth Rate?
![Page 6: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/6.jpg)
Expresión y producción de proteínas
Sistemas de expresión• Bacteriano• Hongos/levaduras• Animales• Plantas• Insectos• Otros
Recuperación de proteínas• Etiquetas de His• Fusiones MBP
![Page 7: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/7.jpg)
Objectivos de la expresión deproteínas en producción industrial
Fermentación segura y bajo control
Barata
Alto nivel de proteína recombinante (g/L nomg/L)
Facilidad de recuperar proteínas expresadas
Que la proteína expresada no se degrade
![Page 8: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/8.jpg)
Huespedes de expresión microbiano – E. coli
VENTAJAS
Gran cantidad de vectores a usa(fago λ phage y plásmidosderivados de pUC)
Altisima eficiencia detransformación
Fermentaciones muy bienconocidas
Recuperación y lisis celular muysencilla
DISVENTAJAS
Problemas de contaminacionescon pirógenos
A veces baja expresión
No glicosilas las proteínas
Proteínas no se exportan
![Page 9: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/9.jpg)
Un vector de E. coli plasmídico
• Cribado por color• promotores de SP6 o T7 RNA polimerasas• Selección de resistencia a Amp
![Page 10: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/10.jpg)
The decision to target the expressedprotein to a specific cellularcompartment, that is, to the cytoplasmicspace, periplasmic space or the culturemedium, rests on balancing theadvantages and disadvantages of eachcompartment
periplasm
cytoplasm
extracellular
[B] Heterologous biological expression systems (in vivo)
1) E. coli
AdvantagesRelatively simple procedureRapid Very high expression level
DisasdvantagesCodon usageSolubility (inclusion body, degeneration)Limitation in the length of cDNALacks post-translational modification(glycosylation, phosphorylation etc.)
Subcellular targeting of expression
Two common promoter systems:- T7 promoter- IPTG (isopropyl thiogalactoside) inducible promoter
+ IPTGLiepman and Olsen (2003)Plant Physiol. 131: 215-227
![Page 11: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/11.jpg)
Huesped de expresión microbiana –Saccharomyces cerevisiae
VENTAJAS
Rendimiento de expresiónmoderado
Glicosilación de Proteínas exp.
Exporta Proteínas exp.
Fermentaciónes muy bienconocidas
Recuperación y lisis celular sencilla
DESVENTAJAS
Eficiencia de transformaciónbaja-media
Un rango limitado de vectores
Genes expresados se debenincorporar de manera estable
![Page 12: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/12.jpg)
Huespedes de espresión microbiana –Pichia pastoris and Kluveromyces sp.
VENTAJAS
Rendimientos deexpresión moderadas
Proteínas glicosiladas
Proteínas exportadas
Recuperación sencilla
DESVENTAJAS
Eficiencia detransformación media-baja
Fermentaciones difíciles
Limitado rango de vectores
Genes de deben deincorporar estable
Uso de codones inusuales
![Page 13: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/13.jpg)
Huespedes de espresión microbiana –Bacillus
VENTAJAS
Rendimientos de expresiónmuy altos (>10 g/L)
Fermentaciones sencillas
Proteínas expoertadas
Sistema de recuperaciónsencilla
Ocurren naturalmente
DESVENTAJAS
Rango limitado de vectores
Los vectores optimizados sonprivados
No glycosila
Sistema de transformaciónvariables
![Page 14: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/14.jpg)
Expresión en células de insecto(Sistema específico huesped-vector; células de insecto en
cultivo Sf9 y vectores de Baculovirus vectors
VENTAJAS Glicosilación
Escalable a volúmenesgrandes
DESVENTAJAS Un sistema de
transformación altamenteespecífico (Baculovirus)
Cultivo fastidioso
Tiempo de crecimiento mylento (tiempo de doblaje es20h)
![Page 15: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/15.jpg)
Cultivo de células de plantas
VENTAJAS Suspensiones celulares
Se requiere baja aireación
Protein dirigidas a lavacuola
DESVENTAJAS Rango restringido de sistemas de
transformación• Agrobacterium para dicots• Biolistica para monocots• Virus de plantas específicos
Crecimiento muy lento (d.t. 15h –48h)
Muy sensible a estrés Crece de manera heterotrófica
pero requiere luz paracrecimiento óptimo
![Page 16: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/16.jpg)
Cultivo de células animales
VENTAJAS Glicosilación exacta
Se pueden tranformar enlineas celulares
Algunas crecen ensuspensiones (p.e. célulasCHO)
Algunas céulas derivadas decarcinoma (p.e. célulasHeLa) crecen muy rápido
Expresión extracelular
DESVENTAJAS La mayoría crecen
adheridas
Requerimientos complejospara crecimiento
Tiempo de crecimiento muylento (t.d. 15-25h)
Muy susceptible acontaminación
Muy sensible a estreses
![Page 17: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/17.jpg)
Otros sistemas de expresión
Sistema de expresión de tabaco Usa TMV Promotores específicos de tejido Se puede escalar
Sistema de expresión en animal completo Transformación de embrion sin fertilizar Uso de promotores específicos de tejidos (p.e. promotor específico
de la lactosa para la expresión sólo en la leche)
![Page 18: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/18.jpg)
Desarrollo de sistemas de expresión
Plásmidos multicopia
Múltiples marcadores de selección (ampicilina,
tetraciclina, kanamicina…)
Promotores diferentes (ITPG, Trp, inducible por
temperatura, Tet)
Multiples secuencias ori
Etiquetas N o C terminal tags (p.e., poli-His) con sitios de
ruptura
![Page 19: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/19.jpg)
Vectores designados para la recuperaciónde proteínas: Etiquetas de His
Vectores contienenpoliGCA
Proteínas tienen polyHis
Recuperación conproteínas de afinidad Ni-NTA
![Page 20: Concept : Cell Yield - UMA...Concept : Cell Yield Glucose, mM 0 50 100 150 200 250 300 0 10 20 30 40 Slope = 7.2 µg/ml per mM Experimental observation – Cell mass is proportional](https://reader035.fdocuments.us/reader035/viewer/2022062603/5f6ef3253bfbc17c2343ed4b/html5/thumbnails/20.jpg)
Recombinant protein expression
[A] Cell free systems (in vitro)
- Rabbit reticulocyte lysate translation system
- Wheat Germ Extract
Contain the cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, and initiation, elongation and termination factors).
- Coupled transcription/translation systems
High protein yields to continuous-exchange cell-free (CECF)technology. A semi-permeable membrane separates twochambers that contain a reaction compartment for coupledtranscription/translation from an enhanced E. coli lysate, and afeeding compartment for substrates and energy components.Protein synthesis can continue for up to 24 hours as substratesand energy components continuously diffuse across the membraneinto the reaction compartment while inhibitory reaction by-productssimultaneously diffuse away, into the feeding compartment. As withother open systems, researchers can add detergents, chaperones,labeled amino acids, cofactors, and protease inhibitors during thereaction and monitor protein quantities at any time. If desired,investigators can co-express multiple proteins by using several DNAtemplates in one reaction