lecture 11 - sequencing long polypeptides

Question 1

list 4 limitations of DNA sequencing

Answer 1

(1) heteromultimers - need to separate into protomers
(2) long sequences
(3) blocked N-term
(4) disulphide bonds

Question 2

how do we sequence long sequences?

Answer 2

• cut protein into smaller fragments using proteases
• cut peptides at a specific sequence
• purify the separate fragments of interest
• sequence each fragment

Question 3

where does trypsin cut

Answer 3

after K/R (pos)

Question 4

where does chymotrypsin cut

Answer 4

after F/Y/W (aromatics)

Question 5

where does V8 protease cut

Answer 5

after D/E (neg)

Question 6

where does Asp-N cut

Answer 6

before D/E (neg)

Question 7

where does CNBr (not enzyme but reagent) cut

Answer 7

after M

Question 8

trypsin/chymotripsin won’t cut before:

why?

Answer 8

proline

can’t fit properly

Question 9

what is the result when trypsin/chymotrypsin/V8/CNBr cuts between two amino acids?

Answer 9

• generate new N-term

- generate a fragment that has amino acid of interest as the C-term

Question 10

what is the result when Asp-N cut between two amino acids?

Answer 10

-generate new fragment where D/E is the 1st residue at the N-term

Question 11

how do we peice together fragments?

Answer 11

• need to use more than one enzyme

- then compare the fragments made by both enzymes for overlapping sequences

Question 12

example question: peice together the fragments

trypsin: FWMGAK, CAQ, LPMDGR
CNBr: GAKLPM, FWM, DGRCAQ

Answer 12

FWMGAKLPMDGRCAQ

Question 13

how do we handle a blocked N-term?

Answer 13

• cleave protein with proteases

- sequence all but the N-term fragment