Class 6 E1

Question 1

What ways can you produce proteins?

Answer 1

1. synthesis in a laboratory (chemistry tool)

2. in a cell (biological tool: cloning)

Question 2

What methods can you use to determine the function of a protein?

Answer 2

protein purification:

1. loss of function
2. gain of function
3. new function.

Question 3

What methods can you use to characterize the properties of a protein?

Answer 3

Conc. and analyze protein in situ / introduce it into cell / tissue / organism via genetic engineering.

Question 4

How does solid-phase peptide synthesis (SPPS) work?

Answer 4

1. growing peptide coupled to insert resin matrix.
2. chemically protected* AA’s added sequentially to growing chain until full length peptide achieved.
3. peptide cleaved from resin.
4. repeat steps for each AA addition.

Question 5

The process of SPPS is?

Answer 5

slow, laborious.

Question 6

What are the caveats of chemical peptide synthesis?

Answer 6

1. best for small peptides.
2. doesn’t include chaperones / modifying enzymes.
3. cellular conditions may be hard to replicate in test tube (type) system.

Question 7

protein overexpression

Answer 7

use of biological system to produce large quantities of folded protein.

Question 8

What is an alternative to SPPS and why?

Answer 8

protein overexpression:

1. larger size range.
2. benefits of other cell components.

Question 9

What does protein overexpression require?

Answer 9

generating piece of DNA that encodes protein and regulatory elements that ensure protein expression.

Question 10

What does protein overexpression use?

Answer 10

an organism / cell to express protein after introducing piece of DNA into cell.

Question 11

expression vectors

Answer 11

DNA that encodes a message for a protein to be expressed and regulatory info needed for that protein’s expression.

Question 12

expression vectors are made via?

Answer 12

fragments of DNA ligated together in lab to combine elements needed.

Question 13

What 4 elements do expression vectors contain?

Answer 13

1. ori seq
2. promoter region
3. cDNA coding seq
4. polyadenylation seq

Question 14

ori seq

Answer 14

origin of replication to produce more plasmids.

Question 15

promoter region

Answer 15

origin of transcription, produce mRNA.

Question 16

cDNA coding seq

Answer 16

seq to encode protein of interest.

Question 17

polyadenylation seq

Answer 17

helps ribosomes hang on longer

Question 18

expression plasmids

Answer 18

tell cells to express.

Question 19

vectors / plasmids are?

Answer 19

circular DNA, contain promoter, selectable marker, elements that enhance expression / detection.

Question 20

expression

Answer 20

DNA to RNA to protein.

Question 21

CMV promoter

Answer 21

viral promoter for gene of interest.

Question 22

hGH poly(A)

Answer 22

viral poly(A) seq for selectable marker gene.

Question 23

multiple cloning site

Answer 23

where you can put your gene of interest.

Question 24

What organisms are used for protein expression?

Answer 24

1. Escherichia coli
2. saccharomyces cerevisiae
3. sf9 cells (army caterpillars)
4. HEK cells

Question 25

site-directed mutagenesis

Answer 25

technique - make small alterations to proteins coding seq via changing DNA.

Question 26

Site-directed mutagenesis mutations generally affect?

Answer 26

single AA at a time.

Question 27

What might you want to consider when designing a site-directed mutagenesis?

Answer 27

1. desired alteration(s) in AA seq.

2. might not get info on all possibilities.

Question 28

Why use site-directed mutagenesis?

Answer 28

identify which NTs and AAs are needed to make a functioning protein.

Question 29

Steps for introducing random mutations to make variants of a protein of interest?

Answer 29

1. source DNA inserted into DNA-cloning vector.
2. perform PCR rxn using mutagenic primers.
3. digest w/ Dpni to cleave original methylated template strands.
4. recover mutated product by introducing it into E.coli by transformation.

Question 30

fusion proteins

Answer 30

generated by in-frame cloning of cDNAs for 2 unrelated proteins / protein domains.

Question 31

transgenes

Answer 31

used to express fusion proteins.

Question 32

Transgenes express fusion proteins by?

Answer 32

fluorescent tags (visual)
protein of interest fused to easily isolated domain or epitope.
enzymes for PCR, lactase, lipases in detergent.

Question 33

What determines the type of fusion protein in an application?

Answer 33

1. where protein is found in cell.
2. what domains of protein bind DNA.
3. which tissues are promoter active in?

Question 34

epitope tags

Answer 34

small domains established w/ isolation procedures; make / isolate a lot of your protein.

Question 35

An epitope tag can serve as?

Answer 35

a label / handle to separate your protein from other cell proteins.

Question 36

fluorescent fusion proteins

Answer 36

in vivo:

GFP, YFP, CFP, RFP.