pET Bacterial Recombinant Protein Vector

1. Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Reaccredited with "A++" Grade by NAAC
K. Narayanapura, Kothanur (PO)
Bengaluru
pET Bacterial Recombinant Protein Vector

2. 1. pET vector is an expression vector used for the expression of recombinant protein in E. coli.
2. The pET vector system is a powerful and widely used system for expressing recombinant proteins in E. coli.
3. The pET vector exists as a low copy number plasmid in host E. coli, which reduces leaky expression before induction.
4. This vector system utilizes the T7 lac promoter system for strong and tightly controlled gene expression
5. In this system, there is a T7 promoter that can be acted upon by T7 RNA polymerase to drive high-level expression of
the gene of interest.
6. Additionally, there is a lac operator (LacO) sequence just downstream of the T7 promoter that can be acted upon by the
lac repressor (LacI) protein to block transcription of the T7 promoter. The plasmid also carries the natural promoter and
coding sequence for LacI. The LacI protein acts to repress expression of the T7 RNA polymerase gene by the host
polymerase.
7. Addition of IPTG blocks the inhibitory action of LacI, thereby inducing expression of T7 RNA polymerase and also
removing LacI inhibition of the gene of interest.
8. The gene of interest is cloned into the pET vector under the control of the strong bacteriophage T7 transcription and
translation regulatory system.
9. Activation of expression is achieved by providing T7 RNA polymerase within the cell.
pET Bacterial Recombinant Protein Vector

3. Although the pET expression system is designed for high-level recombinant protein expression, the expression
level can be reduced by decreasing the amount of IPTG supplied to host cells.
Advantages:
Strong expression: The T7 transcription and translation
regulatory system allows for very high-level production of
proteins of interest, in many cases close to 50% of total
protein in the culture.
Tightly controlled expression: The expression of the
gene of interest is generally very strongly repressed in the
absence of added IPTG, and this “off” state is very robust
for most genes of interest in most host strains.
Vector of Choice
pET-28a(+)
pET-31b(+)
pET-32a(+), pET-32b(+), pET-32c(+)
pET-33b(+)
pET-39b(+), pET-40b(+)
pET-41a(+), pET-41b(+), pET-41c(+) ,pET-
42a(+), pET-42b(+), pET-42c(+)
pET-43.1a(+), pET-43.1b(+), pET-43.1c(+)
pET-44a(+), pET-44b(+), pET-44c(+)
pET-45b(+)
pET-46 Ek/LIC
pET-47b(+), pET-48b(+), pET-49b(+), pET-50b(+)

4. Components:
T7 promoter: Drives high-level transcription of the gene of interest when
T7 RNA polymerase is present. When placed immediately upstream of a
LacO element, the entire cassette is known as the T7lac promoter.
LacO: Binding site for LacI. This element inhibits activity of the T7 promoter
when LacI protein is present, preventing leaky expression of the gene of
interest.
RBS: The ribosome-binding site and translation initiation element from T7
bacteriophage. This allows for efficient production of the protein of interest.
ORF: The open reading frame of your gene of interest is placed here.
T7 terminator: Signal sequence to terminate the transcript made from the
gene of interest, preventing run-on transcription.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be
maintained by ampicillin selection in E. coli.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as
well as the Rop gene exist in low copy numbers in E. coli.
Rop: Repressor of primer. It encodes a small protein that regulates plasmid
copy number. The presence of the Rop protein, in combination of pBR322
origin of replication on the plasmid, results in low copy numbers of the
plasmid.
LacI: The E. coli natural promoter and coding sequence for the lac
repressor. In the absence of induction of the system (i.e. without IPTG), the
LacI protein represses transcription of the gene of interest from the T7lac
promoter, as well as transcription of T7 RNA polymerase from the LacUV5
promoter in host strains used for recombinant protein production.