Recombinant proteins

Question 1

Prokaryote expression systems

Answer 1

Well-characterized systems available
High-level expression easily achieved
Cost-effective

Question 2

Eukaryote expression system

Answer 2

Post-translational modifications may be important for structure and function
(Glycosylation, phosphorylation, intramolecular and intermolecular disulfide bonds)

Question 3

Basic features of a vector

Answer 3

Origin of replication
Selectable marker
Multiple cloning site

Question 4

Special features of a vector

Answer 4

For specific purposes such as Mutagenesis, protein expression

Question 5

Expression vector features

Answer 5

Promoter, terminator, MCS, Ribosome binding site, a sequence for fusion protein or tag

Question 6

Two commonly used origin of replications

Answer 6

ColE! and pUC

Question 7

ColE! origin comes from ___________

Its copy # is _______

Answer 7

plasmid Col E1 and its derivative pBR322 a widely used early cloning vector
low (15-30 copies per cell)

Question 8

pUC-ori is

Answer 8

• the high copy number plasmid pUC (~300)

Question 9

Two diffierent plasmids with the same ori

Answer 9

cannot be maintained in the same cell, they interfere with each others replication

Question 10

Common markers

Answer 10

ampicillan, kanamycin, chloramphenicol, streptomycin, tetracyclin

Question 11

Advantages to inducible or repressible promotoers

Answer 11

Controlled expression of gene prodcuts toxic to host organism,
Control of time and duration of maximal protein expression

Question 12

Common promoters

Answer 12

lac, trp, tac

Question 13

lac promoter

Answer 13

Is a sequence that controls transcription of lac genes,

• it is blocked by a repressor lac1 protein
• induced by IPTG

A repressor protein LacI is continually produced, binds to repressor binding site (operator)

Question 14

trp promoter _______

• blocked by _________
• induced by ___________

Answer 14

Controls transcription of trp genes

• blocked by trptophan
  
  • dimeric TRP aporepressor is bound by two molecules of L-tryptophan forming an active repressor comples
• induced by 3-ß-indoleacrylic acid, which binds more tightly to tryptophan than to the Trp aporepressor, resulting in inactivation of the repressor

Question 15

tac promoter - _________
used in _____
normally _________
_______ acts as an inducer by __________

Answer 15

hybrid of lac and trp promoters (stronger than either)
pGEX vector
LacI binds the lacI binding site and inhibits transcription
IPTG, binding LacI and removing it from the promoter

Question 16

λPʟ Promoter - ___________
blocked by __________
The mutant λc1857 ___________–

Answer 16

controls the transcription of the bacteriophage λ -> strong
[blocked by] λc1 protein
is temperature sensitive (active at 30˚C but not at 42˚C(where the promoter is induced))

Question 17

T7 promoter - _______

Requires __________, of which there are two ways to deliver ________ and ________

Answer 17

Promoter of the bacteriophage T7 RNAP gene
T7 RNAP for expression
Infection with T7 phage
Incorporating T7 RNAP gene into host genome

Question 18

lac1 bacterial operon

Answer 18

Is a group of genes and promoter, plus additional sequences that function together in regulation.
The group of structural genes are transcribed into one polycistronic mRNA

Question 19

IPTG

Answer 19

binds LacI preventing it from binding to the repressor site

Question 20

Rationale between protein and peptide fusions

Answer 20

improve stability and solubility
Facilitate purification procedures (GST-fusion and His-tag)
Provide a sensitive means of detection (GFP and HA tag)
Confer specific localization

Question 21

GST

Answer 21

a family of enzymes that catalyze the transfer of glutathione to substrates
About 220 amino acids, and 26 kDa
Binds glutathione which can be used to purify
fusion proteins
GTS could affect the function of the fusion; can
be removed using a signal peptide for protease
- Factor Xa: cuts after Arg of Ile-Glu/Asp-Gly-Arg
- thrombin: cuts after Arg of Leu-Val-Pro-Arg-Gly-Ser