Lecture 10-11 Flashcards
Bacterial expression vectors:
Promoter sequence
(for the correct sequence)
Promoter allows efficient transcription of the
inserted gene (drives transcription), therefore RNA
binds
Bacterial expression vectors:
Operator sequence
(for the correct sequence)
Operator permits regulation through a specific
repressor that will bind to it
Bacterial expression vectors:
Polylinker
Polylinker with unique sites for several restriction
endonucleases (ie. cloning sitesinsert gene of interest)
○ Polylinker/MCS site of interaction for gene of
interest
Bacterial expression vectors:
Transcription termination sequence
can improve stability
of mRNA and protein yield (minimizes cellular energy
drain)
Bacterial expression vectors:
Selectable genetic marker
(eg. antibiotic resistance)
selectable marker allows selection of cells containing the recombinant expression vector
Bacterial expression vectors:
Origin of replication
a sequence of DNA at which replication is initiated
Bacterial expression vectors:
Gene encoding repressor
Gene encoding repressor that binds to O and regulates P
Bacterial expression vectors:
Ribosome binding site (RBS)
provides sequence signals
required for efficient translation of mRNA derived from the inserted gene.
Bacterial expression vectors:
pGEX- differences?
Ptac promoter: modified promoter to increase binding of RNA polymerase
lacq: mutated lacI gene with higher levels of repressor control of protein expression
Encodes N terminal GST for affinity tag and PreScission for protease cleavage
Bacterial expression vectors:
pET
Pt7: promoter for T7 RNA polymerase
Add IPTG:
-Lac repressor inactivated on both plasmid and bacterial genomic DNA
-T7 RNA pol is produced by host cell RNA pol
-T7 RNA polymerase can now transcribe the gene of interest
= over expression of protein from target gene
Bacterial expression vectors:
Lac operon?
If no lactose is present?
Lac operon: collection of three genes required for lactose transport and metabolism in E.coli
● Adjacent structural genes: lacZ (betagalactosidase), lacY (lactose permease) and lacA (thiogalactoside transacetylase)
If no lactose is present: lac genes are turned off, no enzyme production, therefore energy is saved
Genetic switch turns genes off and on=regulation
Regulation of expression using Lac repressor:
Control mechanism
● Lac repressor (regulatory protein) encoded by lacI gene
● LacI gene is constitutively expressed
● In absence of lactose, Lac repressor binds tightly to
operator sequence
● Lac repressor interferes with binding of RNA polymerase
to promoter
● →prevents gene transcription
Regulation of expression using Lac repressor:
- in presence of lactose (or allolactose) ?
- Consequence?
- Lac repressor cannot bind to operator sequence due to
structural/conformational changes caused by lactose
binding to the repressor protein - Lac repressor no longer interferes with binding of RNA polymerase to promoter
→lacZ, Y and A are transcribed
Induction of protein expression (function of IPTG)
Using the lac repressor in vitro
● can exploit this system for controlled regulation of protein
expression in vitro
● Need to provide:
○ lacI gene plus promoter and operator sequence (expression vector)
● IPTG: bind to lac repressor protein and release from operator, therefore ability to control transcription to regulate lac repressor protein
Optimising translation of proteins
● Need effective translation of proteins from mRNA
transcripts
● If codon usage in the overexpressed protein differs
significantly from the host cell, problems may arise during
protein expression
● Therefore, the expression system needs to maximise the
translation of proteins from this mRNA
Correct processing of proteins:
● Many proteins require the formation of disulphide (SS)
bonds to fold properly
● Without SS bond, proteins may be degraded or precipitate in the cell
Problem:
● expression of proteins occurs in cytoplasm of E.coli
● cytoplasm is a reducing environment that can prevent SS
bond formation
Solution:
Use E.coli strains engineered with mutations in:
● gor glutathione reductase
● trxB thioredoxin reductase
These altered E.coli strains can promote disulphide bond formation and correct folding in the cytoplasm
Affinity tags for protein purification
- What is the basis?
- Used for what?
- Where is it placed?
- Proteins have structural domains: fold independently and have discrete functions
- Can add domains to proteins that can be used to facilitate solubility, folding and enhance purification
- Affinity tag placed where it will not interfere
Affinity tags for protein purification
- 6 X His tag
- MBP
- GST
- 6x His tag: interacts with cobalt and nickel ions
- MBP: interacts with maltose (amylose)
- GST: interacts with reduced glutathione
