lecture 16 - recombinant tech 3

Question 1

why may genes be overexpressed

Answer 1

• protein products important in biotech or medicine
• academic interest

Question 2

what does alpha-antitrypsin treat

Answer 2

emphysema

Question 3

what does epidemal growth factor treat

Answer 3

wound healing

Question 4

what does insulin treat

Answer 4

diabetes

Question 5

what does erythropoietin treat

Answer 5

anemia

Question 6

what does factor vii treat

Answer 6

hemophillia

Question 7

what was factor ix treat

Answer 7

hemophillia

Question 8

what does growth homrone treat

Answer 8

growth disorders

Question 9

what does tissue plasminogen activator treat

Answer 9

heart attacks

Question 10

what does herceptin treat

Answer 10

breast cancer

Question 11

what does enbrel treat

Answer 11

arthritis

Question 12

how are proteins overproduced using E Coli

Answer 12

1. cDNA is cloned into expression vector downstream from strong promoter and ribosome binding site
2. target gene amplified by PCR which inttroduces unique restriction sites at each end
3. often Ndel restriction site is in the multiple cloning side and PCR will create a version of target gene with Ndel site overlapping start codon and restirction site downstream from stop codon (called HindIII site)

Question 13

what do new overproduction systems in e coli use to transcribe rna and why

Answer 13

T7 rna polymerase bc 4x fast than e. coli rna polymerase

Question 14

how can you ensure high yields of protein

Answer 14

bacterial host cells should be grown to high density before foreign gene is induced

Question 15

how do plasmid expression systems control target gene expression

Answer 15

use lacI-encoded repressor (and then induce by adding IPTG which is an analogue of lactose)

Question 16

how are proteins analyzed when they are overproduced

Answer 16

using sds-page

Question 17

what process is SDS-PAGE similar to

Answer 17

gel electrophoresis of nuclei acids

Question 18

what process follows overproduction of proteins

Answer 18

purification of themh

Question 19

why are proteins purified

Answer 19

to free them of contaminants so they can be given therapeutically

Question 20

what slower methods can you use to separate the protein you want from others

Answer 20

ion-exchange chromatography (separate by surface charge)

gel filtration chromatography (separate by size)

Question 21

what faster method can you use to purify proteins

Answer 21

immobilized metal ion affinity chromatography (IMAC)

Question 22

explain IMAC

Answer 22

expression vectors have been developed that allow the target protein to fuse to hexahistidine tag which will bind to nickel atoms so it is the only protein that does not go through. the tag is removed by adding imidazole buffer

Question 23

what are some problems with doing overexpression in e coli

Answer 23

• misfolding (proteins form inactive proteins called inclusion bodies)
• membrane proteins (if too much membrane proteins are made then it can disrupt bilayer and fuck up membrane functions)
• cannot post-translationally modify any eukaryotic proteins that need that

Question 24

explain what erythropoietin is

Answer 24

glycoprotein hormone synetzied by kidney cells which stimulates differentiation of erythrocytes from progenitor cells in bone marrow

Question 25

how is erythropoietin overproduced

Answer 25

overexpressed in chinese hamster ovary from SV40 viral vector

Question 26

what is directed mutagenesis and what is it used for

Answer 26

replacing specific AA residues within polypedtide chains to redesign substrate specificity of enzyme or investigate whether certain side chain is involved in particular protein function

Question 27

explain the quick change method of directed mutagenesis

Answer 27

1. Order synthetic oligonucleotides that are very similar but off by a base or two to switch codon (need to change both strands)
2. Isolate plasmid from e coli that contains DNA adenine methylase that will methylated adenine based within the sequence GATC
3. denature plasmid by heating
4. add in oligonucleotides and amplify dna using PCR
5. use Dpnl (restirction enzyme) to destroy methylated DNA (which is the original DNA) and then only mutated data is left
6. transform into e coli