Post on 11-Jul-2016
description
transcript
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)