Lecture 6

Question 1

True or false: Coding is universal

Answer 1

True

Question 2

What are invariant residues?

Answer 2

positions in the aligned sequences where there is an exact amino acid residue match

Question 3

What are Conservative substitutions

Answer 3

positions in the aligned sequences where match is not exact, similar residues occur

Question 4

Describe identity vs. similarity

Answer 4

percentage of invariant residues between the aligned sequences; percentage of conservative substitutions + invariant residues between the aligned sequences

Question 5

What is cytochrome c, where is it found?

Answer 5

small electron transfer protein that is a member of the mitochondrial respiratory chain - found in eukaryotes as well as a few bacteria

Question 6

Can you use multiple sequence alignments to make a phylogenetic tree?

Answer 6

Yes

Question 7

What does a sequence logo show?

Answer 7

takes the information from a multiple sequence alignment and for each residue number presents the residues that occur most often, with the size of the letter indicating how common that residue occurs there

Question 8

What does it mean for a residue to be highly conserved?

Answer 8

a single residue dominates but is not invariant

Question 9

At position 14, what residue is only shown

Answer 9

Cysteine

Question 10

If two protein sequences are >25% identical, will their structures be different?

Answer 10

No, similar

Question 11

If <25% identical, will their structures be different?

Answer 11

They may still have similar structures

Question 12

Can the fold of the protein be predicted from the sequence alone?

Answer 12

In principle yes, but the ability to do this has not yet been perfected

Question 13

Structure of globin

Answer 13

a protein with a defining fold composed of antiparallel alpha helices that binds to the cofactor heme

Question 14

What does myoglobin do and where is it found

Answer 14

oxygen storage protein that is abundant in the skeletal and heart muscle of mammals where it provides oxygen for the high metabolic needs of these tissues

Question 15

What does hemoglobin do? What does oxygen have to do with its function?

Answer 15

Delivers oxygen to tissues, Hemoglobin has a high affinity for O2 in the lungs and will pick up oxygen, and has a low affinity for O2 in the tissues, where it will release its bound oxygen

Question 16

What does ascaris use its hemoglobin for?

Answer 16

to tightly bind oxygen to block its toxic effect

Question 17

What does nitrogen-fixing bacteria do?

Answer 17

can take nitrogen from the air and convert it to ammonia - readily incorporated into biomolecules.

Question 18

What does nitrogen-fixing bacteria do in a symbiotic relationship?

Answer 18

certain plants surround these bacteria in root nodules
The plant also supplies a globin, leghemoglobin - that tightly binds oxygen and protects the bacterial nitrogenase

Question 19

What do globins do in bacteria?

Answer 19

Oxygen bound to HbN reacts with toxic nitric oxide, NO, produced by the host, to produce harmless nitrate

Question 20

What makes a protein family?

Answer 20

set of proteins with a common fold, Family members have a common evolutionary origin

Question 21

What are the three things that protein engineering includes?

Answer 21

Recombinant protein expression, Protein mutagenesis, Creation of tagged and fusion proteins (chimeric proteins)

Question 22

What is recombinant protein expression?

Answer 22

production of large mounts of a protein from a convenient expression host

Question 23

What is protein mutagenesis?

Answer 23

altering the original sequence (wild type) by targeted changes at specific positions

Question 24

What does the Creation of tagged and fusion proteins (chimeric proteins) do

Answer 24

linking the coding sequences of two proteins to make one larger protein

Question 25

Describe the process of recombinant proteins

Answer 25

DNA sequence that encodes a protein of interest is combined with the DNA sequence of a plasmid vector that will direct high levels of production (expression) of the protein when the vector is introduced into a host such as bacteria

Question 26

What are expression hosts

Answer 26

the organisms that bear the expression vector and which will produce the protein of interest

Question 27

What are expression vectors?

Answer 27

modified plasmids that contain the gene sequence of the protein of interest, and promoters to control its expression

Question 28

What are synthetic oligonucleotides?

Answer 28

short sequences of DNA, synthetically prepared - used as primers in PCR and to introduce targeted mutations into a gene

Question 29

What enzymes are used for manipulation of DNA?

Answer 29

DNA ligases, DNA polymerases, restriction endonuclease

Question 30

What are the four tools for recombinant protein expression?

Answer 30

expression hosts, expression vectors, synthetic oligonucleotides, enzymes for manipulation of DNA

Question 31

What are the pros and cons for using E.coli as a host in recombinant protein expression

Answer 31

Pro: cheap, high yield and easily scalable Con: eukaryotic proteins may not be post-translational processed

Question 32

What are the pros and cons for using yeast as a host in recombinant protein expression

Answer 32

Pro: cheap to grow, high protein yields possible, eukaryotic proteins are post-translationally processed Con: a tough cell wall makes them a pain to break open (lyse) to extract the desired protein

Question 33

Structure of plasmids

Answer 33

circular, double stranded DNA molecules that are found within bacteria

Question 34

What does the origin of replication do?

Answer 34

allows the vector to propagate in the host

Question 35

What does a selectable marker do?

Answer 35

a gene that encodes an antibiotic resistance enzymes so that only host cells that contain the plasmid will grow in culture media that contains the antibiotic

Question 36

What does the multiple cloning site contain

Answer 36

contains unique restriction endonuclease sequences where the vector can be cut to insert the coding sequence for the protein of sequence

Question 37

What do the genetic signals for transcription and translation do?

Answer 37

drive transcription and translation into overdrive so that the recombinant protein is expressed in abundance

Question 38

Explain the five steps in recombinant protein expression

Answer 38

Obtain the target coding sequence (PCR or chemical synthesis of DNA chains), Cut its ends with restriction enzymes (cut DNA at specific sequences), Ligate this to the expression vector, cut with the same restriction enzymes (so that ends of sequence have compatible base pairing), Introduce the expression vector into the host, Host expresses the recombinant protein

Question 39

what is Tissue plasminogen activator (tPA):

Answer 39

a protease that activates plasmin, a protein that dissolves fibrin (clots)

Question 40

What are the four things recombinant protein expression can do?

Answer 40

Large-scale production of valuable proteins, Large scale expression of proteins from rare sources, Creation of tagged proteins for ease of purification and tracking, His6 - tagged recombinant proteins for ease of purification

Question 41

What is a useful modification to add to a DNA sequence for easier purification of the recombinant protein

Answer 41

add a DNA sequence that encodes a stretch of six histidine residues at the N-terminus or C-terminus of the recombinant protein - tagged

Question 42

Explain how His6 tags work

Answer 42

The lone pair of electrons on the imidazole side chain of histidine can coordinate to vacant valence orbitals of a metal ion such as nickel ion

Question 43

Explain the single-step purification of a His-tagged protein

Answer 43

The cell lysate is applied to the NiNTA resin, His-tagged proteins bind, and the rest of the proteins in the lysate are washed off the column with additional buffer - the imidazole compete with the His6-tag for binding the resin