Recombinant proteins Flashcards
Prokaryote expression systems
Well-characterized systems available
High-level expression easily achieved
Cost-effective
Eukaryote expression system
Post-translational modifications may be important for structure and function
(Glycosylation, phosphorylation, intramolecular and intermolecular disulfide bonds)
Basic features of a vector
Origin of replication
Selectable marker
Multiple cloning site
Special features of a vector
For specific purposes such as Mutagenesis, protein expression
Expression vector features
Promoter, terminator, MCS, Ribosome binding site, a sequence for fusion protein or tag
Two commonly used origin of replications
ColE! and pUC
ColE! origin comes from ___________
Its copy # is _______
plasmid Col E1 and its derivative pBR322 a widely used early cloning vector
low (15-30 copies per cell)
pUC-ori is
- the high copy number plasmid pUC (~300)
Two diffierent plasmids with the same ori
cannot be maintained in the same cell, they interfere with each others replication
Common markers
ampicillan, kanamycin, chloramphenicol, streptomycin, tetracyclin
Advantages to inducible or repressible promotoers
Controlled expression of gene prodcuts toxic to host organism,
Control of time and duration of maximal protein expression
Common promoters
lac, trp, tac
lac promoter
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)
trp promoter _______
- blocked by _________
- induced by ___________
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
tac promoter - _________
used in _____
normally _________
_______ acts as an inducer by __________
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
λPʟ Promoter - ___________
blocked by __________
The mutant λc1857 ___________–
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))
T7 promoter - _______
Requires __________, of which there are two ways to deliver ________ and ________
Promoter of the bacteriophage T7 RNAP gene
T7 RNAP for expression
Infection with T7 phage
Incorporating T7 RNAP gene into host genome
lac1 bacterial operon
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
IPTG
binds LacI preventing it from binding to the repressor site
Rationale between protein and peptide fusions
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
GST
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
