29. Applications of Recombinant Technology

Question 1

what is one important reason we want to clone a gene (focus of this lecture)

Answer 1

protein (the gene product) for profit

Question 2

what are two examples of recombinant proteins that help human diseases

Answer 2

pituatry dwarfism - human growth hormone used to be used.

diabetes - pig insulin which could cause immune responses

now recombinant proteins from bacteria used

Question 3

what is an expression vector?

Answer 3

the vector (ie plasmid) that carries the gene of interest that can be expressed into a protein

Question 4

what are four important parts for a sucessful expression vector?

Answer 4

1. it expresses into protein
2. soluble in solution
3. able to be detected
4. can be purified

Question 5

what are 3 probelms of expression in eukaryote cells?

Answer 5

1. the protein produced is foreign - gets degraded by proteases
2. proteins have disulfide bridges - must have them in expressed proteins too
3. incorrect fold in protein which can cause insoluble inclusion bodies

Question 6

what is perplasmic space?

Answer 6

the space in between the two layers of membranes in gram negative bacteria cells

Question 7

why is perplasmic space important for cloning DNA for proteins? 3 points

Answer 7

1. is less reducing so disulfide bridges can form in prtoeins
2. no proteases present - dosent remove foreign protein
3. easy to purify

Question 8

how can protein go to the periplasm when its already in the cytoplasm?

what are the three steps for this to occur?

Answer 8

via passage through a membrane protein on the membrane in e-coli.

1. signal sequence targets protein to membrane
2. signal sequence celaves as protein is exported
3. protein released into periplasm space of bacetera