expression vectors

1. Expression vectors

2. DEFINITION
• The expression vector, otherwise known as
an expression construct, is usually
a plasmid or virus designed for protein
expression in cellsThe expression vector is
a plasmid engineered to introduce a
particular gene into the target cell.

3. • Expression vectors must have expression signals
such as a strong promoter, a strong termination
codon, adjustment of the distance between the
promoter and the cloned gene, and the insertion
of a transcription termination sequence and
a portable translation initiation sequence.
• Expression vectors are used for molecular biology
techniques such as site-directed mutagenesis.

4. Introduction
• Once the expression vector is inside the cell, the protein
that is encoded by the gene is produced by the cellular-
transcription and translation machinery ribosomal
complexes.
• The plasmid is frequently engineered to contain regulatory
sequences that act as enhancer and promoter regions and
lead to efficient transcription of the gene carried on the
expression vector.
• The goal of a well-designed expression vector is the
production of large amounts of stable messenger RNA, and
in extension, proteins. Expression vectors are basic tools
for biotechnology and the production of proteins such as
insulin, which is important for the treatment of diabetes.

5. • After expression of the gene product, the purification of the protein is
required; but since the vector is introduced to a host cell, the protein of
interest should be purified from the proteins of the host cell. Therefore, to
make the purification process easy, the cloned gene should have a tag.
This tag could be histidine (His) tag or any other marker peptide.
• Expression vectors are used for molecular biology techniques such as site-
directed mutagenesis. Cloning vectors, which are very similar to
expression vectors, involve the same process of introducing a new gene
into a plasmid, but the plasmid is then added into bacteria for
replication purposes. In general, DNA vectors that are used in many
molecular-biology gene-cloning experiments need not result in the
expression of a protein.
• Expression vectors must have expression signals such as a strong
promoter, a strong termination codon, adjustment of the distance
between the promoter and the cloned gene, and the insertion of a
transcription termination sequence and a PTIS (portable translation
initiation sequence).

6. • Expression vectors produce proteins through the transcription of the
vector's insert followed by translation of the mRNA produced, they
therefore require more components than the simpler transcription-only
vectors. Expression in different host organism would require different
elements, although they share similar requirements, for example a
promoter for initiation of transcription, a ribosomal binding site for
translation initiation, and termination signals.
• Prokaryotes expression vector
• Promoter - commonly used inducible promoters are promoters derived
from lac operon and the T7 promoter. A stronger
promoter; Trp/Tryptophan Operon and Tac Promoter, a hybrid collection
of both the Trp and Lac Operon promoters.
• Ribosome Binding Site (RBS) Follows the promoter, and promotes efficient
translation of the protein of interest.
• Translation initiation site - Shine-Dalgarno sequence enclosed in the RBS,
8 base-pairs upstream of the AUG start codon.

7. • The basic requirement for any expression
system is a promoter cloning site(s) next to it
and transcriptional terminator.
• Origin suitable for replication initiation in a
particular bacteria or any host.
• A selection marker gene such as antibiotic
resistance gene.
• Regulator elements.
• Shine Delgarno sequence.

9. pLac-Z expression vectors

10. • Lac –Z promoter operator is in frame with lac-Z
alpha fragment (the NH3 terminal part of
Galactosidase gene.
• Multiple cloning sites are found in the border of
NH3 end including ATG sequence.
• The presence of such restriction site sequences
should not disturb the functional activity of the
protein, which complements with the omega
fragment of the Lac-Z produced by the bacterial
cell as the complement.
• If any gene is placed in proper frame in the MCS
the protein expressed will be is fused form.
• The expression of the gene can be regulated by
IPTG (Isopropyl thio b-Galactoside).

11. pET Expression Vector:

12. • The size of the vector is 5700bp
• It has T7 promoter adjacent to lac operator.
• Next to it is a sequence called Shine Delgarno
sequence.
• Adjacent to S/D sequence there are few cloning sites
such as Nde I, Nhe I and BamH I, at the end of which is
T7 phage transcriptional terminator is found.
• For the expression of this gene the bacterial cell
should provide T7 RNA polymerase, which is under the
control of Lac-Z operator/promoter.
• The repressor produced by the bacterial lac-IQ can be
regulated by IPTG.