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