Protein expression Flashcards
why might someone choose euk expression vs prok?
can get post-trans mods
what are the feature vectors>
Basic features
• Origin of replication (ori) • Selectable marker
• Multiple cloning site
Special features
• For specific purposes such as – Mutagenesis
– Protein expression
what are the feature expression vectors?
- Promoter
- Terminator
- Multiple cloning site
- Ribosome binding site
- Asequenceforfusionproteinor“tag”
common oris and there copy #s?
ColE1 and pUC– 15-30 and 300 copies per cell
Incompatibility:two different plasmids using the same ori cannot be maintained in the same cell. They interfere with each other’s replication
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describe kanamycin MoA and subsequent resolvation
inhibits 30S ribosome; phopshorylated to kanamycin-P via neomycin phosphotransferase II
compare inducible and repressible promoter
inducible–gene normally off; add inducer to induce gene expression
repressible–gene normally on; and repressor to prevent induction
commonly used promoters and
• lacpromoter:sequencethatcontrols transcription of lac genes; blocked by a repressor LacI protein, but induced by IPTG (isopropylthiogalactoside)
• trp promoter: sequence that controls transcription of trp genes; blocked by tryptophan, but induced by 3-β-indoleacrylic acid–IAA binds Trp aporepressor, inactivates–>transcription
• tac promoter : a hybrid of lac and trp promoters; stronger than either, inducible by IPTG–binds inhibitor LacI
• λPL promoter: controls the transcription of the bacteriophage λ → strong
- Blocked by λcI protein
- temperature sensitive mutant λcI857, which is active at 30oC, but inactive at 42oC (promoter is induced)
• T7promoter
- promoter of bacteriophage T7 DNA-dependent RNA polymerase (RNAP) gene
- Requires T7 RNAP for expression
purpose of protein fusion?
• Improvestabilityorsolubility
• Facilitate purification procedures
- GST fusion (glutathione-conj’d agarose)–GST v soluble; can also be used to bind column (pruification)
- His-tag (metal chelation chromatography)
• Provide sensitive means of detection - GFP
- HA-tag
• Conferspecificlocalization
• GTScouldaffectthefunctionofthefusion;can be removed using a signal peptide for protease
- Factor Xa: cuts after Arg of Ile-Glu/Asp-Gly-Arg
- thrombin: cuts after Arg of Leu-Val-Pro-Arg-Gly-Ser
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GFP fusion
• 236 amino acids and about 27 kDa
• Hasrevolutionizedhowwestudyprotein localization and functions in cells; GFP fusion proteins can be visualized directly
• VariousGFPvariantsallowmultipleproteinsto be observed simultaneously
• Also,antibodytoGFPallowsdetectionofthe fusion protein by Western blotting
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short polypeptide tags–purify by fusing, peptides bind to Abs; good for high affinity
ya
study slide 29
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general steps of expression in bacteria
- Insert coding sequence into MCS
- Transform bacterial host & selection (take into account codon bias of species–solved by mutagenesis)
- (Induce) high level protein production
- Protein purification
what factors influence levels of protein expression?
• Promoter strength: frequency of transcription initiation
• Plasmid copy number: # of templates
• Codonbias:availabilityoftRNAspeciesin host organism
• Protein stability and recovery
– Degradation of foreign proteins
– Accumulation as insoluble proteins in bacterial “inclusion bodies”
large DNA frags can be cloned into yeast
ya
Promoters for yeast expression
- Should be tightly regulated, thus allowing the expression of proteins toxic to the cell
- Should have a high induction ratio to background noise
- Several are used and the most widely used
is GAL1 promoter
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describe gal yeast promoter/induction
galactose induces inducer via gal3–>inducer binds to gal 80; therefore, with no gal 80 binding to gal 4, gal 4 is free to activate GAL genes (is a transcription factor)
markers for yeast expression?
Markers:Genesinvolvedinthebiosynthesisof amino acids are often used. Cell lines would carry the recessive mutant, while expression vectors would carry a wild type gene, allowing positive auxotrophic selection
- His3 - Leu2 - Trp1 - Ura3
Selection on a medium lacking the particular amino acid
why might we express proteins in mammalian cells?
Reasons
• For mammalian genes, it is an environment closer to the native environment – fewer problems in expression
• Proteinsarefoldedintothenativeform
• Allow the correct post-translational modifications
• Proteins can be secreted, allowing isolation of proteins from the medium
what are the components of a mammalian plasmid vector?
Plasmid vectors • Origin of replication (ori) • Selectablemarker • Multiple cloning site • Forstrongandproperexpression - Promoter - Terminator (polyadenylation signal) - Intron
what are the oris in mammalian plasmids?
• SV40 ori: from simian virus 40, which causes lytic infection
- Very high copy number in transfected cells - Good for transient expression
- Low frequency of stable transfection
• BK or BPV ori: from human BK and bovine papilomas virus, which cause latent infections
- low to moderate copy
- Plasmid DNA using the ori is maintained in cells autonomously and stably for a long time
• EBV ori: from human Epstein-Barr virus
- Replicon is even more stable than BK and BPV ori
selectble markers in mammalian expression vectors?
• Endogenous mutant genes as markers
- mutant causes a defect which can be compensated by the wild type gene
- Marker is recessive and can only be used in the mutant cell lines; thus limited use
• Dominant markers
- Confer a phenotype that is novel to the cell - Usually drug resistance
what is geneticin?
an antibiotic which inhibits elongation of protein synth in both proks and euks
