Recombinant protein
expression.
Other alternatives
Elvira Marín – Felipe Clemente
WG1 - Protein expression UCM
La Cristalera – Miraflores – 10/12/12
Unkown proteins cloned
Chromosome 16
̴ 260 Unkown proteins
Cloned in pANT7_cGST
Dr. Manuel Fuentes
WG1
25 new proteins
Digest with
restriction enzymes
Mass spectrometry
(MRM)
Sequencing
Expression (IVTT) and
purification (GST)
Unkown proteins UN-CLONED
Chromosome 16
Unkown proteins
Cloned in pANT7_cGST
Design primers
Gateway cloning system
Master clone
pDONR221
(recombinase)
Unkown proteins uncloned
pANT7_cGST
(recombinase)
Digestion and
Sequencing
Mass spectrometry
(MRM)
Expression (IVTT) and
purification (GST)
Protein expression
Protein expression systems
Prokaryotes
Bacteria
Escherichia coli
Eukaryotes
Yeast
Saccharomyces cerevisiae
Pichia pastoris
Mammalian
CHO, HeLa, BHK
Protein expression systems
LOW
HIGH
SPEED
Mammalian
Yeast
Bacteria
COST
Bacteria
Yeast
Mammalian
YIELD
Mammalian
POSTTRANSLACTION
MODIFICATION
Bacteria
Bacteria
Yeast
Yeast
Mammalian
Protein expression systems
N.I. I M
E. coli
Yeast
Mammalian
E. coli compartments
Advantages
Simple purification
Proteolysis is less extensive
Improved folding (S-S
formation)
Disadvantages
Signal does not always
facilitate export
Inclusion bodies may be
formed
Periplasm
Inclusion bodies:
easy purification
protection from proteases
Cytoplasm
inactive protein (non-toxic)
Higher protein yield (until
30% Biomass)
Simpler plasmid construct
Less extensive proteolysis
Extracellular Simple purification
Improved folding
Inclusion bodies:
protein folding
denaturation/refolding
Usually no secretion
Cell lysis
Fusion partners to the recombinant protein
Maltose binding
protein (MBP)
GlutathioneS-transferase
(GST)
N-utilization
substrate (NusA)
Small ubiquitinmodifier (SUMO)
Thioredoxin
Pichia pastoris vs Saccharomyces cerevisiae
Advantages P. pastoris and S. cerevisiae
Short doubling time
Readily manipulated genome
Improved folding and post-translational modification
Expression of similar genes and compatible vectors
P. pastoris
Better yield of recombinant protein (higher cell density)
Methylotrophic yeast (methanol as its only carbon source)
Strongly methanol induced promoters
(alcohol oxidase genes: AOX1 and AOX2)
Optimal growth pH 3.0-7.0
Extremely low levels of endogenous protein secretion
Expression vectors integrated in the genome
Disulfide bond formation and glycosylation modifications
S. cerevisiae
Expression on mammalian cells
All posttranslational modifications
Highest folding capacity (antibodies…)
Secreted recombinant proteins for an easier
purification rather than intracellular production
Lowest yield
Transfection more complex than plasmid
transformation
Stable or transient transfection (takes
longer to obtain stable transformants)
Protein expression systems summary
Bacteria
Yeast
Advantages
Disadvantages
Fastest expression method (days)
Inexpensive bioproduction media and
high density biomass
Simple process scale-up
Well characterized genetics
Limited posttranslational modifications
Unsoluble proteins and not correctly
folded
Rapid expression method (weeks)
Inexpensive bioproduction media and
high density biomass
Most posttranslational modifications
High folding capacity
N-linked glycan structures different
from mammalian forms
Transient-transfection
Mammalian
Moderate rapid expression method (weeks)
All posttranslational modifications and
high folding capacity
Low density biomass and expensive
bioproduction media
Difficult process scale-up
Stable-transfection
Mammalian
All posttranslational modifications and
high folding capacity
Low density biomass and expensive
bioproduction media
Difficult process scale-up
Longest expression method (months)
Elvira Marín – Felipe Clemente
WG1 - Protein expression UCM
Dra. Concha Gil
Dr. Manuel Fuentes
E. coli expression systems
N.I. I M