1.

The following is the plasmid map/sequence for plasmid pET-30a(+) from Novagen (company
name).

Please select correct answer(s) for the following scenarios according to the pET-30a plasmid
information:

a) Scenario 1: If you want to use this pET-30a plasmid to overexpress a foreign gene containing
Nde I and Sal I restriction sites at its 5- and 3-end, the overexpressed protein will have (5
marks)
(A) One His-tag and S-tag
(B) One His-tag
(C) S-tag (D) Two His-tags
b) Scenario 2: If you know that His-tag would affect the target protein function or activity after
purification, hence the target overexpressed protein should not contain any His-tags at its
terminals, which of the following restriction site(s) must be part of the DNA insert? (5 marks)
(A) Xba I (B) Nde I or Xba I (C) Nde I (D) Bgl II (E) Xho I (F) Bgl II or Xho I (G) Nsp V
c) Scenario 3: Enterokinase can cleave off the His-tag from His-tagged proteins. If you plan to
cleave off the N-terminal His-tag of an overexpressed protein after immobilized metal
affinity chromatography step, which of the following restriction enzyme combination(s) can
be chosen when you clone the DNA insert into pET-30a plasmid, provided your choice would
not digest the DNA insert? (5 marks)
(A) Bgl II and Nco I (B) Kpn I and Nco I (C) Nco I and Hind III

(D) Xho I and EcoR I

(E) EcoR V and Hind III (F) Not I and Nco I (G) EcoR V and Nde I (H) Xba I and Bgl II