Programma modulo C

•Espressione•Purificazione•Mutagenesi•Evoluzione assistita

Ingegneria proteica: espressione, purificazione e manipolazione di proteine ricombinanti

E1 – Ottimizzazione espressione proteine eterologhe

•Il sistema d’espressione pEt•Efficienza di traduzione•Stabilità prodotto finale•Corpi di inclusione•Ottimizzazione processo di purificazione

Protein production for biotechnological applications

•Enzymes•Vaccines•Antibodies•Protein chips•Biosensors•Structural and functional studies•Drug development•Search for interactors

Protein Expression

1. Transcriptional Efficiency

2. Translational Efficiency

3. Stability of the final product

DNA RNA Protein

(Strategies for efficient production of heterologous proteins in Escherichia coli. Jana S, Deb JK. Appl Microbiol Biotechnol. 2005. 67, 289-98. )

Transcriptional Efficiency

• Promoter sequence

• Terminator (stem-loop structure)

Overexpression of heterologous proteins in E. Coli by the Novagen pEt system

•The strongest promoters are those from viruses and bacteriophages

•It would be good to exploit them for the expression of recombinant proteins

•However, bacterial cells don’t have the RNA polymerase that can recognize them...

Overexpression of heterologous proteins in E. Coli by the Novagen pEt system

Advantage inducible systems

Uncouple cell growth and protein production

pEt systems are particularly little “leaky” (well repressed in the absence of IPTG)

Translational Efficiency

• mRNA Stability

• Shine-Dalgarno Sequence

• Codon Bias (codon usage)

•Le cellule usano i codoni sinonimi con diversa frequenza

•I codoni usati con maggiore frequenza corrispondono ai tRNA più abbondanti

•I codoni “preferiti” sono usati nei geni altamente espressi, gli altri sono usati nei geni poco espressi

•Le preferenze per codoni sinonimi sono diverse in organismi diversi!

•Con codon usage sfavorevole si ha bassa efficienza di traduzione e alto rischio di frameshift

Codon Usage in E. coli & humans

Possible strategies to overcome codon-usage problems

• Optimized E. coli cells (Rosetta) overexpressing tRNA for 7 rare codons

• Site-directed mutagenesis

Protein stability

Possible strategies to overcome protein instability problems

• Controlling protein localization to avoid accumulation in the cytoplasm

- B. Subtilis (Gram positive) => medium- E. Coli (Gram negative) => periplasm

• Coexpression of chaperons

Secretion-based strategies

Advantages:

Protection from degradationOxidizing envirormentIndependence of toxicityEnriched

Promoter/operator Leader peptide Target gene

Disadvantages:

DilutedTranslocation efficiency

Correct formation of disulfide bridges

Oxidizing envirorment may not be enough

=> Foldases DsbA (disulfide oxidoreductase) and DsbC (disulfide bond isomerase) in the E. Coli periplasm

=> In some cases effective co-expression of foldases

Inclusion bodiesAround 50% of heterologous proteins overexpressed in E. coli form inclusion bodies

Advantages

Enrichment by centrifugationProtection from proteasesHigh-level accumulation

Disadvantages

ResolubilizationRefolding

Procedure

1000-10000 g8M urea or 6M GdmGl (DTT)Dialysis

Prevention

Lower temperatureWeaker promoterFusion proteins

Over-expression often results in misfolding, or accumulation at folding intermediate states - proteins accumulation as insoluble aggregates – INCLUSION BODIES.

Reduction of bacterial growth temperature (following induction) from 37 °C to 30 °C or 25 °C can significantly reduce inclusion body formation.

Inclusion bodies are very dense – they sediment very readily by low-speed centrifugation performed immediately after cell homogenisation. Inclusion bodies sediment more rapidly than the cell debris.

Denaturants – urea, guanidinium chloride, detergents, organic solvents, alkaline pH.Denaturant removed by dialysis, dilution or diafiltration - refolding

MBP/Mat a1

linker

Amount and purity

Industrial production moles-mmoles 99%Crystallization/X-ray μmoles 95%NMR μmoles 95%Functional studies nmoles variableAntibody production nmoles 90%Sequencing pmoles 90%Mass spectrometry fmoles even quite low

Assay•Specific•Rapid•Sensitive•Quantitative

•Enzymatic activity•Biological function•Binding activity•Antibody...

For overexpressed proteins, SDS gels in the absence of functional assay (but dangerous at later stages...)

How things usually don’t work...

Properties/Methods

Solubility Ammonium-sulfate, PEG precipitationSize/shape Gel filtration, UltracentrifugationpI (charge) Ion exchange, IsoelectrofocusingHydrophobicityReversed phaseBinding Affinity techniquesStability Thermal precipitation

An interesting approachto protein purificationis to make it pure in first place...

Up to mg yield

Cell-freeprotein

expressionsystems

http://www.roche-applied-science.com/sis/proteinexpression/literature/manual/cell_free.htm

Also available extracts from eukaryotic cells

Protein recovery by 1 step of affinity chromatography

Possible to use linear DNA directly from PCR amplification

Main advantages of cell-free protein expression

•High throughput protein production

•Efficient production of proteins difficult to express in vivo (toxic, with disulfide bridges, that form inclusion bodies, prone to proteolytical degradation etc.)

•Efficient production of membrane proteins thanks to the addition of lipids/detergents

•Possibility to modify by natural or unnatural post-translational modifications (e.g. incorporation of fluorofores)

•Possibility to introduce unnatural amino acids (engineered aminoacyl-tRNA synthetases to mischarge tRNA)

•Possibility to produce higher-order assemblies (e.g. viral capsid for vaccines)

(Approfondimento: He 2008 New Biotechnology 25, 126-132)