EXPRESSION VECTORS

• The expression vector is usually a plasmid or virus designed for

protein expression in cells.
• It is a plasmid engineered to introduce a particular gene into the target
cell.
• They must have expression signals such as strong promoter, strong
termination codon, adjustment of the distance between the promoter
and the cloned gene, and the insertion of transcription termination
sequence and portable translation initiation sequences.
• They are used for molecular biology techniques such as site directed
mutagenesis.
• Molecular cloning and genetics studies require the use of vectors as a host
or carrier system to transfer selected genetic components, namely DNA, to
cells or organisms.
• Specialized host vectors are available for cloning and expression of a
variety of genes from numerous species and of variable size fragments.
• Vector technology facilitates library construction, single gene analysis,
protein expression, and basic molecular cloning.
• The selected vector system is influenced by downstream applications,
such as PCR, gene transfection or gene transduction.
• Plasmids and viral vectors are two of the most common vector types used.
• The development of fluorescence protein marked vectors, like GFP (Green
Fluorescent Protein), allows the detection of gene expression within live
cells.
• Antibiotics resistance, localized expression, engineered selection markers
and cloning site size should be taken into consideration when choosing an
appropriate vector
 HOST
 Expression vector used have specific
elements for a particular organisms.
• BACTERIAL
- Rapid and cheap production .
- E.coli and Bacillus subtilis .
- lac operator and T7 promoter.
- Ex pGEX series .
• YEAST
- Saccharomyces cerevisiae , pischia
pastoris.
- Hybrid system.
• PLANT
- Agrobacterium tumefaciens .
- T- DNA sequence, 25kb, selectable marker
• MAMMALIAN
- Folding of product
- Post translational modifications
- Specific activity of enymes
- Adenoviral vectors, vaccinia vectors, retroviral
vectors and baculovirus.
 EXAMPLES
1. pET vector
• Size 5700 bp
• These are a family of expression vectors that
utilise phage T7 promoters to regulate synthesis
of cloned gene products.
• It is engineered to take advantage of the
features of T7 bacteriophage gene 10 that
promote high level transcription and translation.
• It is one of the most widely used systems for
the cloning and in vivo expression of
recombinant proteins in E.coli.
2. pLac Z vector
• Lac –Z promoter operator is in frame with lac-Z alpha fragment (the
NH3 terminal part of Galactosidase gene.
• Multiple cloning sites are found .
• 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)
3. pTac vector
This has the combination of Trp and Lac-Z promoter –operator elements,
which is inducible.
It is considered to be a very efficient promoter.
 PURIFICATION TAGS
Peptide sequences that are attached to proteins to facilitate easy
detection and purification of expressed proteins.
They can also be used to identify potential binding partners for your
protein of interest.
There is a wide variety of protein tags that can be used to suit your
purpose.
a) Affinity Tags
purify proteins from crude cell lysates. Affinity tagged proteins can
be purified by using a specifc affinity resin & antibodies. Some of
the most commonly used affinity tags include the following:
• Glutathione-S transferase (GST): GST can be used to purify a
protein by inserting its coding sequence next to your protein of
interest. They will then be expressed as a fusion protein after
transcription and translation.. This technique can be used to
explain direct protein-protein interactions.
• Poly-Histidine tag (His): This tag comprises of 6-8 histidine residues.
The relatively small size of His tags makes its integration into
expression vectors extremely easy. His-tagged proteins are purified
using immobilized nickel, cobalt or zinc ions, and eluted using EDTA or
imidazole.

• Calmodulin Binding Protein (CBP): The relatively small size of CBP (4

kDa) makes it ideal for purifying delicate proteins under mild
conditions. The tag binds to a calmodulin resin and the proteins can be
eluted with a neutral buffer containing low concentrations of EGTA, a
calcium chelator.

• Maltose-binding protein (MBP): These tags bind to amylase agarose

and are commonly used to increase the solubility of fusion proteins.
b) Epitope Tags
They are typically smaller than affinity tags and are readily recognized
by antibodies. Due to their relatively small size, they have extremely
little or no effect on the structure of the resulting fusion protein.
• Myc tag – This tag is a short peptide sequence (EQKLISEEDL )
derived from the c-myc gene product and recognized by numerous
commercial antibodies. for isolating protein complexes with
multiple subunits.
• FLAG tag – Like the Myc tag, the FLAG tag is a popular short
peptide tag (DYKDDDDK) for isolating protein complexes with
multiple subunits.
c) Fluorescent Tags
Due to their non-toxic nature, these tags can be used to detect tagged
proteins in both live and fixed cells.
Green fluorescent protein (GFP) is one of the most widely used protein
tags under this category. GFP is a protein isolated from the jellyfish
Aequorea victoria that exhibits bright green fluorescence that does
not fade easily when exposed to blue or ultraviolet (UV) light.