Week 4: Protein Sequence Info Flashcards
What are 3 implications of common genetic code?
- Evidence of life’s unity 2, molecular evolution 3. recombinant protein expression and engineering.
Describe evolution of myoglobin.
Myoglobin - 153 residues - sequence comparable b/w mammals - few conservative and nonconservative subs.
What is an invariant residue?
Invariant residues - positions in aligned sequences where exact residue match
What is a conservative substitution
similar but not exact
What is identity?
percent of invariant residues b/w sequences
What is similarity?
percentage of conservative subs and invariant residues between aligned sequences.
How are invariant and variant subs displayed?
*= invariant, : = conserved sub
What do multiple sequence alignments show?
reveal evolutionary relationships.
What does it mean if two protein sequences are > 25% identical?
They will have similar structures
What does it mean if two protein sequences are < 25% identical
may still have similar structures as fold is more highly conserved than sequence.
What does sequence determine?
Fold !
Describe myoglobin
Oxygen storage
Describe hemoglobin
Oxygen transport
Describe Ascaris globin
protects parasitic intestinal worm from oxygen toxicity
Describe leghemoglobin
protects bacterial nitrogenase in root nodules of plants
What is a protein family?
common fold ie. globins. Common evolutionary origin. Different, specific functions from differences in amino acid sequences among members.
What are some types of protein engineering?
recombinant protein expression, protein mutagenesis, creation of tagged protein and fusion proteins (chimeric proteins).
How is protein engineering possible?
- The genetic code is universal. 2. Molecular biologists and organic chemists have provided the tools.
How are recombinant proteins made? What tools are used?
Put DNA sequence into bacteria to create recombinant human DNA expressed in bacteria.
Tools: Expression hosts, vectors, enzymes for DNA manipulation, synthetic DNA (oligonucleotide primers for PCR, full-length genes).
Describe pros and cons of using E. coli and yeast as recombinant protein hosts?
E.coli - Pro: cheap, high yields, easily scalable. Con - no post-translational processing of eukaryotic proteins
Yeast - Pro: cheap, high yields, eukaryotic proteins are post-translationally processed. Con: tough to lyse the cells.
Describe recombinant protein vectors
Plasmids. Have Ori, selectable marker (ie. antibiotic resistance, cloning site where gene is put and signals to increase gene expression
Describe the 5 steps of creating recombinant proteins + how to verify.
- Obtain target coding sequence (PCR or chemical synthesis of DNA chains)
- Cut ends with restriction enzymes
- Ligate this to expression vector, cut with the same restriction enzymes
- Introduce the expression vector into the host
- Host expresses the recombinant protein
Use gel electrophoresis to verify
What are 3 uses of protein engineering?
- Large-Scale Production of Valuable Proteins - ie. tissue plasminogen activator- activates plasmin - a serine protease dissolves fibrin from clots - actilyse is recombinant tPA.
- Large Scale Expression of Proteins from Rare Sources - ie. fossils
- Creation of Tagged Proteins for Ease of Purification and Tracking - ie. histidine tag $His_6$. Lone pair on imidazole side chain of His can coordinate to vacant valence orbitals of metal ion ie. nickel. A string of 6 multiples the effect. Using a chelating resin with nickel bound to it ie. nickel(II) nitroloacetate-agarose. Then elute with imidazole.
