MS LECTURE 2 VECTORS Flashcards
Expression vectors have to?
Facilitate expression (transcription and translation) of the inserted open reading frame
Expression vectors have to contain
a promoter region upstream of the inserted gene
product (to facilitate transcription)
* have to contain other elements required for proper transcription and translation of the gene (e.g. transcription termination sites, translation
initiation sites)
These have to be matched with the cell system they are used for.
pET system
Inducible bacterial expression plasmid
T7 promoter under the control of
lac operator
T7 being under lac operator control ensures that
expression of our inserted gene product can be regulated.
T7 promoter is usually repressed by
lac repressor protein, but can be de-repressed by adding IPTG (isopropyl-beta-D-thiogalactopyranoside).
Why use inducible promoters?
Some recombinant proteins are toxic to the cells and
kill them or stop their growth.
Protein quality can suffer, e.g. by aggregation and/or
degradation when large amounts of protein are
produced in a cell.
Making protein costs energy, regulation ensures protein isn’t produced unnecessarily
Induce protein production (by adding IPTG) when cells are in
early/mid log growth phase.
Protein production is monitored by
determining the optical density (OD) of the bacterial
culture with a spectrophotometer (the more bacteria, the cloudier the
culture).
Regulation of protein expression in the lac operon: without inducer
Repressor binds to operator, preventing transcription of lac operon
Regulation of protein expression in the lac operon with the presence of an inducer
Allolactose binds to repressor and prevents its binding to the operator, meaning that the genes can be transcribed. In the lab IPTG is added which is a non-hydrolysable analogue of allolactose, to de-repress genes under the control of the lac operator
Regulatory elements can be used to
drive expression of other genes e.g. GOI in pET
Genes involved in lactose metabolism
B-galactosidase, permease, transacetylase
T7 promoter is transcribed by
T7 RNA polymerase, a a gene encoded by the T7 phage. High transcription rate. Means the T7 RNA polymerase also needs to be introduced into E. coli as it does not naturally occur in these cells
Genetically modified E. coli strain used for protein expression
E. coli strain BL21/DE3
E coli strain BL21/DE3
Inducible expression of T7 RNA polymerase (under control of lac operon).
T7 RNA polymerase gene and elements of the lac operon have been introduced into the bacterial chromosome.
Addition of IPTG de-represses both, the T7 RNA polymerase and the gene of interest in pET plasmid.
BL21/DE3 strain is also deficient in ompT and lon proteases.
Inducible bacterial expression vectors (like pET) in BL21/DE3 E.coli absence of inducer
In the absence of IPTG, repressor binds to lac operator and transcription is switched off. Lac operator controls
expression of T7 polymerase in BL21/DE3 E.coli strain and expression of insulin gene in pRSET.
Inducible bacterial expression vectors (like pET) in BL21/DE3 E.coli presence of inducer
IPTG binds to repressor and renders it unable to
binds to lac operator. Transcription is unblocked. T7 polymerase is expressed in BL21/DE3 strain and can access T7 promoter in pRSET vector. Lac operator in pRSET is also unblocked by IPTG.
Expression of proteins in mammalian cells
We can introduce plasmid vectors into mammalian cells
through:
- transfection (e.g. Calcium phosphate-mediated or
liposome-mediated)
Or
- transduction with viral vectors.
Features of mammalian expression vectors
You have to be able to amplify these plasmids in E.coli before use, so they still
contain a bacterial replication origin and antibiotic resistance gene (here
Kanamycin). Vectors that work in two different systems are called ‘shuttle
vectors’ (e.g. E.coli/mammalian or E.coli/Yeast).
SV40 origin allows episomal replication in cells lines that contain Simian Virus
40 (SV40) large T antigen (e.g. HEK293T cells). This leads to increased
plasmid numbers in the mammalian cells and therefore higher expression levels.
Gene of interest under control of mammalian or viral promoter (e.g. CMV
promoter from Cytomegalovirus, cellular EF-1a or Ubiquitin C promoters), the
promoter should mediate high level constitutive expression.
SV40 poly A: transcription termination site, adds poly-A tail to mRNA
Different vectors for different purposes;
For expressing fusion proteins (tag sequence contained in vector) :
- Containing affinity or epitope tags: His, GST, Ha, c-myc, flag…
The epitope tags allow you to purify and/or detect your protein
(e.g. affinity purification or immunoprecipitation using antibodies
directed against epitope tag, Western Blot). - Containing fluorescent protein tags (e.g. green fluorescent
protein, GFP). Allows you to visualise your fusion protein in cells,
organisms).
Different vectors for different purposes
Inducible expression:
Tet on/Tet off system (expression only in the presence/absence of
tetracyclin). Similar idea to lac operon/lac promoter system, but
engineered for mammalian system.
Affinity tags:
6x Histidine (His-tag)
Glutathione-S-transferase (GST-tag)
—->
Affinity-tagged
proteins can be
purified via affinity
chromatography
Epitope tags:
Epitope-tagged
proteins detected with
antibodies raised to
these epitopes. They
can also be
‘immunoprecipitated’
using these antibodies
coupled to a solid
matrix.
