Introduction to pET, pcDNA and CMV expression vectors. Critical components of these vectors. Functions of different components

1. Critical components of pET, pcDNA and CMV expression system
Characteristic features of pEt expression vector
 The pET expression vectors are derived from the pBR322 plasmid.
 They are used for protein expression in E. coli. A number of variants of
host cells are available for various cloning and expression needs.
 Target genes are cloned in pET plasmids under control of strong
bacteriophage T7 transcription and (optionally) translation signals;
expression is induced by providing a source of T7 RNA polymerase in the
host cell.
 T7 RNA polymerase is so selective and active that almost all of the cell’s
resources are converted to target gene expression; the desired product can
comprise more than 50% of the total cell protein a few hours after
induction.
 Target genes are initially cloned using hosts that do not contain the T7
RNA polymerase gene, thus eliminating plasmid instability due to the
production of proteins potentially toxic to the host cell.
 Once established in a nonexpression host, plasmids are then transferred
into expression hosts containing a chromosomal copy of the T7 RNA
polymerase gene under lacUV5 control, and expression is induced by the
addition of IPTG.
 In addition to the T7 promoter, all the vectors contain the gene 10 5´ leader,
which facilitates highly efficient translation.

2.  The protein coding sequence of interest may be cloned directly after the
gene 10 initiation codon using the Nde I (pET-3,-11, a, b and c) or Nco I
sites (pET-3d, and -11d).
 Alternatively, the pET-3 and pET-11 vectors contain BamH I cloning sites
in all three reading frames relative to the gene 10 reading frame.
 The gene 10 transcription terminator is also included downstream of the
cloning sites to allow efficient termination of transcription.
 In order to provide adequate levels of lacI protein to shut off T7
polymerase expression as well as T7 promoter transcription, the lacI gene
is included on the pET-11 plasmids.
 All of the vectors in the pET-3 and pET-11 series contain the -lactamase
gene for ampicillin resistance.
Characteristic features of pcDNA3 expression vectors
pcDNA3 vectors are mammalian expression vectors.

3. Characteristic features of pcDNA3 expression vectors
S.N. Feature Benefit
1. Human
cytomegalovirus
(CMV)
immediate-early
promoter/
enhancer
Permits efficient, high-level expression of target
recombinant protein
2. T7 promoter/priming
site
Allows for in vitro transcription in the sense
orientation and sequencing through the insert
3. SP6 promoter SP6 prokaryotic promoter allows for in vitro
transcription in reverse orientation
4. Multiple cloning site in
forward or reverse
orientation
Allows insertion of target gene and facilitates
cloning
5. Bovine growth
hormone (BGH)
polyadenylation signal
Efficient transcription termination and
polyadenylation of mRNA
6. f1 origin Allows rescue of single-stranded DNA
7. SV40 origin and early
promoter
Allows efficient, high-level expression of the
neomycin resistance gene and episomal replication
in cells expressing SV40 large T antigen

4. 8. ColE1 origin High-copy number replication and growth in E. coli
9. SV40 early
polyadenylation signal
Efficient transcription termination and
polyadenylation of mRNA
10. Neomycin resistance
gene
Selection of stable transfectants in mammalian cells
11. Ampicillin resistance
gene (β-lactamase)
Selection of vector in E. coli
12. Ampicillin resistance
gene (β-lactamase)
Allows selection of transformants in E. coli
Characteristic Features of CMV expression System
Characteristic features of CMV expression system
S.N. Feature Benefit
1. CMV immediate-early
promoter
Permits efficient, high-level expression of target
recombinant protein
2. Bovine growth
hormone (BGH)
polyadenylation signal
Efficient transcription termination and
polyadenylation of mRNA
3. SV40 origin SV40 origin for replication in mammalian cells
expressing SV40 Tantigen
4. pBR322 origin Allows efficient, high-level expression in
prokaryotic cells

5. 5. (MCS) Multiple
cloning site
Allows insertion of target gene and facilitates
cloning
6. Ampicillin resistance
gene (β-lactamase)
Selection of vector
7. f1 origin For single-stranded DNA production
Figure: Cloning in an expression vector