recombinant DNA technology

1. Submitted by-
KABERI NATH
ROLL NO-17PBT206

2.  Vectors with strong, controllable promoters are used to maximize the synthesis of cloned
gene products.
 Generally, cloned gene preceded by a promoter recognized by the host cell leads to detectable
synthesis of the cloned gene product.
 But recombinant DNA technology is mainly used for synthesis of large quantities of protein,
either to study its properties or because it has commercial value.
 To maximize gene expression only selecting the strongest promoter possible is not enough, as
the effects of overexpression on the host cell also need to be considered.
 Overexpression of a protein may be toxic to the host cell.
 High-level synthesis can also exert metabolic drain on the cell leading to slower growth.
 To minimize the problems associated with high-level expression, it is usual to use a vector in
which the cloned gene is under the control of a regulated promoter.
2

3. Contd………
 pET - Plasmid for Expression by T7 RNA polymerase
 Originally constructed by Studier and colleagues.
 Size 5700 bp.
 These are a family of expression vectors that utilize phage T7 promoters to
regulate synthesis of cloned gene products.
 Derived from the pBR322 plasmid, pET vectors engineered to take advantage of
the features of the 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.
3

4.  pET vector expression system usually consist of-
1. Site of transcription with lac operon and gene of interest
2. Origin of replication and antibiotic resistance gene
3. lacI for production of Lac operon repressor protein
 Normal function- no protein expression
(LacI protein represses transcription by blocking T7 RNA polymerase
expression)
 Altered function- protein expression
(IPTG binds to Lac repressor protein and expresses T7 RNA polymerase for
transcription) 4

5.  The gene for T7 RNA polymerase (gene 1) is inserted into the chromosome of E. coli and
transcribed from the lac promoter; therefore, it will be expressed only if the inducer
IPTG is added.
 The T7 RNA polymerase will then recognise the T7 promoter on the vector and
transcribe the gene cloned into the pET vector.
 If the protein product of the cloned gene is toxic, it may be necessary to further reduce
the transcription of the cloned gene before induction.
 The T7 lysozyme encoded by a compatible plasmid, pLysS, will bind to any residual T7
RNA polymerase made in the absence of induction and inactivate it.
 Also, the presence of lac operators between the T7 promoter and the cloned gene will
further reduce transcription of the cloned gene in the absence of the inducer IPTG.
5

6. Fig: pET vector expression system
6

7.  The T7 promoter is one of the strongest known promoters. It can produce a lot of protein.
 The pET plasmids have many common restriction sites, especially in front of the T7 promoter
but also in other places.
 The very strong T7 promoter is regulated by the lac operator. In addition, the plasmids encode
their own lac repressor which reduces the leakiness of the promoter.
 The pET plasmids have a medium copy number. (~20-25 per cell), which can be helpful
because it prevents weird things from happening due to copy numbers that are too high or too
low.
 It allows for the high expression level of the T7 promoter without overloading the cell with
many copies of the plasmid in addition.
7

8. Despite the strong selectivity of the T7 promoter for its phage-encoded
polymerase, residual "leaky" expression of very toxic proteins from the basic pET
constructs can be sometimes lethal to the cell.
8

9.  The pET System is the most powerful system yet developed for the cloning and
expression of recombinant proteins in E. coli.
 The pET System provides six possible vector-host combinations that enable tuning
of basal expression levels to optimize target gene expression. These options are
necessary because no single strategy or condition is suitable for every target
protein.
9

10. 1. S.B. Primrose and R.M. Twyman. “Principles of Gene Manipulation and
Genomics”. Seventh Edition
2. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. “Use of T7 RNA
polymerase to direct expression of cloned genes”. Methods Enzymol.
1990;185:60-89
3. Studier FW, Moffatt BA. “Use of bacteriophage T7 RNA polymerase to direct
selective high-level expression of cloned genes”. J Mol Biol. 1986 May
5;189(1):113-30.
4. Dubendorff JW, Studier FW. "Controlling basal expression in an inducible T7
expression system by blocking the target T7 promoter with lac repressor".
Journal of Molecular Biology. 1991 ;219 (1): 45–59.
10

11. 11