Week 4: Protein Sequence Info

Question 1

What are 3 implications of common genetic code?

Answer 1

1. Evidence of life’s unity 2, molecular evolution 3. recombinant protein expression and engineering.

Question 2

Describe evolution of myoglobin.

Answer 2

Myoglobin - 153 residues - sequence comparable b/w mammals - few conservative and nonconservative subs.

Question 3

What is an invariant residue?

Answer 3

Invariant residues - positions in aligned sequences where exact residue match

Question 4

What is a conservative substitution

Answer 4

similar but not exact

Question 5

What is identity?

Answer 5

percent of invariant residues b/w sequences

Question 6

What is similarity?

Answer 6

percentage of conservative subs and invariant residues between aligned sequences.

Question 7

How are invariant and variant subs displayed?

Answer 7

*= invariant, : = conserved sub

Question 8

What do multiple sequence alignments show?

Answer 8

reveal evolutionary relationships.

Question 9

What does it mean if two protein sequences are > 25% identical?

Answer 9

They will have similar structures

Question 10

What does it mean if two protein sequences are < 25% identical

Answer 10

may still have similar structures as fold is more highly conserved than sequence.

Question 11

What does sequence determine?

Answer 11

Fold !

Question 12

Describe myoglobin

Answer 12

Oxygen storage

Question 13

Describe hemoglobin

Answer 13

Oxygen transport

Question 14

Describe Ascaris globin

Answer 14

protects parasitic intestinal worm from oxygen toxicity

Question 15

Describe leghemoglobin

Answer 15

protects bacterial nitrogenase in root nodules of plants

Question 16

What is a protein family?

Answer 16

common fold ie. globins. Common evolutionary origin. Different, specific functions from differences in amino acid sequences among members.

Question 17

What are some types of protein engineering?

Answer 17

recombinant protein expression, protein mutagenesis, creation of tagged protein and fusion proteins (chimeric proteins).

Question 18

How is protein engineering possible?

Answer 18

1. The genetic code is universal. 2. Molecular biologists and organic chemists have provided the tools.

Question 19

How are recombinant proteins made? What tools are used?

Answer 19

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).

Question 20

Describe pros and cons of using E. coli and yeast as recombinant protein hosts?

Answer 20

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.

Question 21

Describe recombinant protein vectors

Answer 21

Plasmids. Have Ori, selectable marker (ie. antibiotic resistance, cloning site where gene is put and signals to increase gene expression

Question 22

Describe the 5 steps of creating recombinant proteins + how to verify.

Answer 22

1. Obtain target coding sequence (PCR or chemical synthesis of DNA chains)
2. Cut ends with restriction enzymes
3. Ligate this to expression vector, cut with the same restriction enzymes
4. Introduce the expression vector into the host
5. Host expresses the recombinant protein

Use gel electrophoresis to verify

Question 23

What are 3 uses of protein engineering?

Answer 23

1. Large-Scale Production of Valuable Proteins - ie. tissue plasminogen activator- activates plasmin - a serine protease dissolves fibrin from clots - actilyse is recombinant tPA.
2. Large Scale Expression of Proteins from Rare Sources - ie. fossils
3. 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.