recombinant protein expression. other alternatives elvira marín – felipe clemente wg1 - protein...
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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_cGSTDr. Manuel Fuentes
WG125 new proteins
Digest with restriction enzymes Sequencing
Expression (IVTT) and purification (GST)
Mass spectrometry(MRM)
Unkown proteins UN-CLONED
Chromosome 16 Unkown proteins
Design primers
Cloned in pANT7_cGST
Gateway cloning system
pDONR221 (recombinase)
Master clone
Unkown proteins uncloned
Expression (IVTT) and purification (GST)
Mass spectrometry(MRM)
Digestion and Sequencing
pANT7_cGST
(recombinase)
Protein expression
Protein expression systems
Yeast
Prokaryotes
Bacteria Mammalian
Eukaryotes
Escherichia coli Saccharomyces cerevisiaePichia pastoris
CHO, HeLa, BHK
Protein expression systems
SPEED
COST
YIELD
POST-TRANSLACTION MODIFICATION
LOW HIGH
Bacteria
Yeast
Yeast
Yeast
Yeast
Bacteria
Bacteria
Bacteria Mammalian
Mammalian
Mammalian
Mammalian
E. coli Yeast Mammalian
Protein expression systems
N.I. I M
Periplasm
Cytoplasm
Extracellular
Advantages
Simple purificationProteolysis is less extensiveImproved folding (S-S formation)
Disadvantages
Signal does not always facilitate exportInclusion bodies may be formed
Inclusion bodies: easy purification protection from proteases inactive protein (non-toxic)Higher protein yield (until 30% Biomass)Simpler plasmid construct
Inclusion bodies: protein folding denaturation/refolding
Less extensive proteolysisSimple purificationImproved folding
Usually no secretion Cell lysis
E. coli compartments
Fusion partners to the recombinant protein
Small ubiquitin-modifier (SUMO)
Glutathione-S-transferase
(GST)
Thioredoxin
N-utilization substrate (NusA)
Maltose binding protein (MBP)
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
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
P. pastoris
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)
Advantages
Fastest expression method (days)Inexpensive bioproduction media andhigh density biomassSimple process scale-upWell characterized genetics
Limited posttranslational modificationsUnsoluble proteins and not correctly folded
Protein expression systems summary
Yeast
Bacteria
Mammalian
Disadvantages
Rapid expression method (weeks)Inexpensive bioproduction media andhigh density biomassMost posttranslational modificationsHigh folding capacity
N-linked glycan structures different from mammalian forms
Transient-transfection
Moderate rapid expression method (weeks)All posttranslational modifications and high folding capacity
Low density biomass and expensivebioproduction mediaDifficult process scale-up
Stable-transfection
Low density biomass and expensivebioproduction mediaDifficult process scale-upLongest expression method (months)
All posttranslational modifications and high folding capacityMammalian
Elvira Marín – Felipe Clemente WG1 - Protein expression UCM
Dra. Concha GilDr. Manuel Fuentes
N.I. I M
E. coli expression systems