L17 Recombinant Protein Expression

Question 1

Why is recombinant protein expression used in purification?

Answer 1

it facilitates easier purification

Question 2

Why is recombinant protein expression helpful in structural biology?

Answer 2

it produces large quantities of protein for crystallography

Question 3

Name a therapeutic use of recombinant protein expression.

Answer 3

Producing vaccines, insulin, or growth hormones

Question 4

How does recombinant expression help study elusive proteins?

Answer 4

It enables the production of proteins not easily extracted from natural sources.

Question 5

What makes E.coli a cheap host for exression?

Answer 5

Low cost of culture and maintenance.

Question 6

Why is E.coli considered a safe expression host?

Answer 6

It doesn’t contain human pathogens

Question 7

What makes E.coli generally easy to manipulate?

Answer 7

it has a single, fully sequenced chromosome

Question 8

Why are mutated E.coli strains useful for tracking proteins?

Answer 8

They require labelled amino acids from the medium

Question 9

What is a limitation of E.coli in expressing human proteins?

Answer 9

It lacks post-translational modifications

Question 10

What common protein modifications can’t E.coli perform?

Answer 10

Glycosylation, disulphide bonds, phosphorylation, and proper folding

Question 11

What is codon bias in E.coli?

Answer 11

it lacks tRNAs for rare human codons like AGA

Question 12

What is the prokaryotic -35 consensus promoter sequence?

Answer 12

TTGACA

Question 13

What is the -10 promoter sequence in prokaryotes?

Answer 13

TATAAT

Question 14

What is the ribosome binding site in E.coli called?

Answer 14

Shine-Dalgarno sequence ( short nucleotide sequence found in the 5’ untranslated region (UTR) of bacterial mRNA, acting as a ribosome binding site)

Question 15

What is the sequence of E.coli’s Shine-Dalgarno site?

Answer 15

AGGAGGU

Question 16

Where is the sequence of E.coli’s Shine-Dalgarno sequence located?

Answer 16

~8 bases upstream of the start codon

Question 17

What is the start codon for translation initiation?

Answer 17

ATG

Question 18

What kind of promoters are preferred in expression vectors?

Answer 18

Inducible promoters

Question 19

What is a fusion partner used for?

Answer 19

Tagging or aiding solubility and purification

Question 20

What is the role of the terminator in expression vectors?

Answer 20

to signal the end of transcription

Question 21

Name two inducible promoters used in bacteria.

Answer 21

lac and tac promoters

Question 22

What induces the lac promoter?

Answer 22

IPTG or allolactose

Question 23

What happens if ribosome binding site spacing is incorrect?

Answer 23

expression efficiency is reduced

Question 24

What is a selectable marker in an expression vector?

Answer 24

antibiotic resistance gene

Question 25

Why are low copy number vectors often used?

Answer 25

to avoid toxicity from overexpression

Question 26

What organism does T7 bacteriophage infect?

Answer 26

E.coli

Question 27

What enzyme drives transcription in the T7 system?

Answer 27

T7 RNA polymerase

Question 28

What regulates the expression of T7 RNA polymerase?

Answer 28

the lac promoter

Question 29

What molecule is used to induce T7 expression?

Answer 29

IPTG

Question 30

Why is the T7 system highly productive?

Answer 30

T7 RNA polymerase is more active in E.coli than RNA polymerase

Question 31

What is a tag in protein expression?

Answer 31

a short peptide added for purification or detection

Question 32

What must be true for fusion proteins to be translated properly?

Answer 32

they must be in the same reading frame

Question 33

What is needed to add a tag to the N-terminus?

Answer 33

the tag gene is added before the gene of interest

Question 34

What is done to add a tag to the C-terminus?

Answer 34

remove the stop codon, add tag, then new stop codon

Question 35

Name a commonly used purification tag.

Answer 35

6His Tag

Question 36

What is GST used for?

Answer 36

affinity purification using glutathione

Question 37

What tag is smaller than GST and interferes less?

Answer 37

HA tag

Question 38

What fusion protein allows visual tracking in cells?

Answer 38

green fluorescent protein (GFP)

Question 39

What does SUMO fusion help improve?

Answer 39

protein solubility

Question 40

What resistance marker does pET SUMO use?

Answer 40

kanamycin resistance gene

Question 41

What is the promoter used in pET SUMO?

Answer 41

T7 promoter

Question 42

What tag is included in the pET SUMO vector?

Answer 42

6His-SUMO

Question 43

What enables gene insertion in pET SUMO?

Answer 43

overhanging Ts

Question 44

What organism produces the Hepatitis vaccine Hecolin?

Answer 44

E.coli

Question 45

What is the first step in affinity purification?

Answer 45

overexpress gene of interest with a fusion tag

Question 46

What column is used to purify GST fusion proteins?

Answer 46

Glutathione-coated column

Question 47

What enzyme digests GST-tagged proteins for purification?

Answer 47

Thrombin

Question 48

What is the second purification step called

Answer 48

Reverse affinity purification

Question 49

What is required when co-transforming with multiple plasmids?

Answer 49

Plasmid compatibility.

Question 50

What system uses two promoters in one vector?

Answer 50

dual promoter system

Question 51

What is a bicistronic vector?

Answer 51

one promoter, two ribosome binding sites, two genes

Question 52

What limits expression in bicistronic systems?

Answer 52

lower expression of downstream genes

Question 53

What is the key difference in introducing DNA to eukaryotic cells?

Answer 53

Transfection, not transformation

Question 54

What expression system is used for the HPV vaccine?

Answer 54

yeast or insect cells

Question 55

What is an advantage of yeast systems?

Answer 55

high yield and eukaryotic protein modification

Question 56

What makes mammalian cells ideal for human protein production?

Answer 56

Correct folding and modifications

Question 57

What is the risk of using mammalian cells?

Answer 57

contamination with animal viruses

Question 58

What viral vector is used in insect expression systems?

Answer 58

Baculovirus

Question 59

What is the expression promoter in baculovirus systems?

Answer 59

pPolh promoter

Question 60

What is a key benefit of cell-free expression systems?

Answer 60

fast and allows toxic protein expression