蛋白结构域互作分析方法
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• In vivo - express fusion protein in vivo
– Purify complexes from the cell
• In vitro - overexpress protein in vitro
– Bind fusion protein to a column and run whole cell lysate through the column. Identify proteins that “stick” to the fusion protein.
– Many times fusions will not be functional.
• Quality of the antibody.
– Is it good enough to precipitate enough protein for analysis?
Computational methods
– GAL4, LexA
• Protein you are studying = Bait
– Fused to the DNA binding domain of GAL4
• Protein(s) you are screening = Fish or Prey
– Fused to the activation domain of GAL4
– Rosetta Stone – Co-regulation – Phylogenetic analysis
Yeast 2-hybrid approach
• Based on the fact some transcriptional activators have separable DNA binding (BD) and transcriptional activation domains (AD).
Hirano et al, 1997 Cell, Vol 89, 511-521, 16 May 1997
Fusion protein affinity chromatography
• Express the protein of interest as a fusion protein.
– 6-8X His residues – Glutathione S-transferase (GST) – Other “tags”
• No longer undergoing reductive evolution.
– Normally would find pseudogenes - not found.
Protein complexes - why?
• Proteins often function as large, multisubunit complexes.
Yeast 2-hybrid on a genome wide scale
• Clone every gene in your genome into both the “bait” and “fish” vectors. • Systematically screen each gene for interactions.
• Genome is quite compact.
– 95% of genome codes for genes. 552.
• Not primitive.
– Has complete set of information pathway and cell cycle genes found in archaea.
Yeast 2-hybrid
• False-positives
– Some baits are “sticky” leading to non-functional interactions
• False negatives
– Binding not tight enough to detect interaction – Fusion proteins often do not fold correctly
• Bind and purify the protein of interest
– Poly His residues will bind Ni2+ – GST will bind glutathione
Image from: Sigma-Aldrich
Fusion proteins - identifying interactions.
• Rosetta stone analysis
– Search for proteins that are separate in one organism but are fused into one protein in another organism.
Computational methods
• N. equitans lacks genes necessary for many aspects of central metabolism.
– Can’t make lipids, vitamins, amino acids, etc. – Parasite, not symbiont? First archaea
• Works best when comparing two proteins suspected of interacting • Bacterial 2-hybrid systems
Co-immunoprecipitation
• Using an antibody to isolate and purify a protein from a whole cell lysate. • Normally you will only purify the protein the antibody recognizes. • Any additional proteins that co-purify are candidates for interacting proteins.
Phylogenetic analysis
• Search for the presence of a protein in all organisms. • Determine the distribution. • Identify other proteins that also show this distribution. • Functionally interact? Physically?
• Obligate symbiont with Ignicoccus • Smallest completely sequenced genome
– <500kB – Genome reduction observed in symbionts (Schmidt) – Is N. equitans a “primitive” archaea or is the genome undergoing reductive evolution?
ຫໍສະໝຸດ Baidu
Difficulties when using biochemical approaches
• Stability of protein:protein interactions.
– Many are not stable enough to survive purification.
• Is the fusion protein functional?
• Co-expression
– Genes that are in operons are often functionally linked. (not always true). – Determine if the structure of an operon is conserved, indicating co-expression. – Candidates for interaction. – Not a great method.
– ribosomes
• Can get clues about the function of a protein by knowing what other proteins it contacts.
Protein:protein interactions
• Genetic approach
– Yeast 2-hybrid
• Transform Bait and Fish plasmids into yeast, measure the expression of a reporter gene.
– Usually a gene can be selected for when expressed.
Image from: http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
• Biochemical approach
– Co-immunoprecipitation – Fusion protein affinity chromatography
• Cell-biology
– FRET - fluorescence resonance energy transfer
• Computational
PLEX
• Protein Link EXplorer. • Uses phylogenetic profiles to predict possible associations.
– Mate individual yeast strains.
• Many false positives.
Interactome
Term to define all of the protein interactions that take place in the cell. Book example - predicting human interactions. Based on data that only 10% of the measured interactions are physiological
• Protein domains vs. structure domains - an example.
Genome of the week
• Nanoarchaeum equitans - archaea
– Hyperthermophile – Diverged early in evolution from other archaea – New kingdom of archaea?
– Purify complexes from the cell
• In vitro - overexpress protein in vitro
– Bind fusion protein to a column and run whole cell lysate through the column. Identify proteins that “stick” to the fusion protein.
– Many times fusions will not be functional.
• Quality of the antibody.
– Is it good enough to precipitate enough protein for analysis?
Computational methods
– GAL4, LexA
• Protein you are studying = Bait
– Fused to the DNA binding domain of GAL4
• Protein(s) you are screening = Fish or Prey
– Fused to the activation domain of GAL4
– Rosetta Stone – Co-regulation – Phylogenetic analysis
Yeast 2-hybrid approach
• Based on the fact some transcriptional activators have separable DNA binding (BD) and transcriptional activation domains (AD).
Hirano et al, 1997 Cell, Vol 89, 511-521, 16 May 1997
Fusion protein affinity chromatography
• Express the protein of interest as a fusion protein.
– 6-8X His residues – Glutathione S-transferase (GST) – Other “tags”
• No longer undergoing reductive evolution.
– Normally would find pseudogenes - not found.
Protein complexes - why?
• Proteins often function as large, multisubunit complexes.
Yeast 2-hybrid on a genome wide scale
• Clone every gene in your genome into both the “bait” and “fish” vectors. • Systematically screen each gene for interactions.
• Genome is quite compact.
– 95% of genome codes for genes. 552.
• Not primitive.
– Has complete set of information pathway and cell cycle genes found in archaea.
Yeast 2-hybrid
• False-positives
– Some baits are “sticky” leading to non-functional interactions
• False negatives
– Binding not tight enough to detect interaction – Fusion proteins often do not fold correctly
• Bind and purify the protein of interest
– Poly His residues will bind Ni2+ – GST will bind glutathione
Image from: Sigma-Aldrich
Fusion proteins - identifying interactions.
• Rosetta stone analysis
– Search for proteins that are separate in one organism but are fused into one protein in another organism.
Computational methods
• N. equitans lacks genes necessary for many aspects of central metabolism.
– Can’t make lipids, vitamins, amino acids, etc. – Parasite, not symbiont? First archaea
• Works best when comparing two proteins suspected of interacting • Bacterial 2-hybrid systems
Co-immunoprecipitation
• Using an antibody to isolate and purify a protein from a whole cell lysate. • Normally you will only purify the protein the antibody recognizes. • Any additional proteins that co-purify are candidates for interacting proteins.
Phylogenetic analysis
• Search for the presence of a protein in all organisms. • Determine the distribution. • Identify other proteins that also show this distribution. • Functionally interact? Physically?
• Obligate symbiont with Ignicoccus • Smallest completely sequenced genome
– <500kB – Genome reduction observed in symbionts (Schmidt) – Is N. equitans a “primitive” archaea or is the genome undergoing reductive evolution?
ຫໍສະໝຸດ Baidu
Difficulties when using biochemical approaches
• Stability of protein:protein interactions.
– Many are not stable enough to survive purification.
• Is the fusion protein functional?
• Co-expression
– Genes that are in operons are often functionally linked. (not always true). – Determine if the structure of an operon is conserved, indicating co-expression. – Candidates for interaction. – Not a great method.
– ribosomes
• Can get clues about the function of a protein by knowing what other proteins it contacts.
Protein:protein interactions
• Genetic approach
– Yeast 2-hybrid
• Transform Bait and Fish plasmids into yeast, measure the expression of a reporter gene.
– Usually a gene can be selected for when expressed.
Image from: http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
• Biochemical approach
– Co-immunoprecipitation – Fusion protein affinity chromatography
• Cell-biology
– FRET - fluorescence resonance energy transfer
• Computational
PLEX
• Protein Link EXplorer. • Uses phylogenetic profiles to predict possible associations.
– Mate individual yeast strains.
• Many false positives.
Interactome
Term to define all of the protein interactions that take place in the cell. Book example - predicting human interactions. Based on data that only 10% of the measured interactions are physiological
• Protein domains vs. structure domains - an example.
Genome of the week
• Nanoarchaeum equitans - archaea
– Hyperthermophile – Diverged early in evolution from other archaea – New kingdom of archaea?