pET vector and T7 expression system

The pET vector system is a powerful and widely used system for expressing
recombinant proteins in E. coli. The gene of interest is cloned into the pET vector
under the control of the strong bacteriophage T7 transcription and translation
regulatory system. Activation of expression is achieved by providing T7 RNA
polymerase within the cell. When the system is fully induced, nearly all of the cell’s
resources are devoted to expressing the gene of interest. With just a few hours of
induction, the recombinant protein could comprise nearly half of the cell’s total
protein.

The T7 expression system is used in the field of microbiology to clone recombinant

DNA using strains of E. coli.[1] It is the most popular system for expressing
recombinant proteins in E. coli.[2]

By 2021, this system had been described in over 220,000 research publications.[3]

Development

The sequencing and annotating of the genome of the T7 bacteriophage took place in
the 1980s at the U.S. Department of Energy's Brookhaven National Laboratory, under
the senior biophysicist F. William Studier. Soon, the lab was able to clone the T7
RNA polymerase and use it, along with the powerful T7 promoter, to transcribe
copious amounts of almost any gene.[4] The development of the T7 expression
system has been considered the most successful biotechnology tool developed at the
Brookhaven National Laboratory, being licensed by over 900 companies which has
generated over $55 million for the lab.[5]

Mechanism

An expression vector, most commonly the pET expression vector, is engineered to

integrate two essential components: a T7 promoter and a gene of interest downstream
of the promoter and under its control. The expression vector is transformed into one
of several relevant strains of E. coli, most frequently BL21(DE3). The E. coli cell also
has its own chromosome, which possesses a gene that is expressed to produce T7
RNA polymerase. (This polymerase originates from the T7 phage,
a bacteriophage virus which infects E. coli bacterial cells and is capable of integrating
its DNA into the host DNA, as well as overriding its cellular machinery to produce
more copies of itself.) T7 RNA polymerase is responsible for beginning transcription
at the T7 promoter of the transformed vector. The T7 gene is itself under the control
of a lac promoter. Normally, both the lac promoter and the T7 promoter are repressed
in the E. coli cell by the Lac repressor. In order to initiate transcription,
an inducer must bind to the lac repressor and prevent it from inhibiting the gene
expression of the T7 gene. Once this happens, the gene can be normally transcribed to
produce T7 RNA polymerase. T7 RNA polymerase, in turn, can bind to the T7
promoter on the expression vector and begin transcribing its downstream gene of
interest. To stimulate this process, the inducer IPTG can be added to the system. IPTG
is a reagent which mimics the structure of allolactose, and can therefore bind to the
lac repressor and prevent it from inhibiting gene expression. Once enough IPTG is
added, the T7 gene is normally transcribed and so transcription of the gene of interest
downstream of the T7 promoter also begins.[6] Expression of a recombinant protein
under the control of the T7 promoter is 8x faster than protein expression under the
control of E. coli RNA polymerase.[7] Basal levels of expression of T7 RNA
polymerase in the cell are also inhibited by the bacteriophage T7 lysozyme, which
results in a delay of the accumulation of T7 RNA polymerase until after lysozymic
activity is saturated.[8]
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.

Disadvantages
Host requirements: Completed pET vectors should be maintained in an E. coli strain
lacking the T7 RNA polymerase gene, and must be transferred to a separate host
strain containing the T7 RNA polymerase gene before induction of protein expression.

Potential leaky expression in some hosts: Even in the absence of IPTG, there may
be some low-level expression of T7 RNA polymerase from the LacUV5 promoter in
some expression host strains, which could lead to bacterial toxicity for certain genes
of interest in certain host strains.

Application

During the COVID-19 pandemic, mRNA vaccines have been developed

by Moderna and Pfizer to combat the spread of the virus. Both Moderna and Pfizer
have relied on the T7 expression system to generate the large quantities of mRNA
needed to manufacture the vaccines.[9][4]

Video guide:

https://www.youtube.com/watch?v=IZXdbLxjr2M

https://www.youtube.com/watch?v=_J9noI2UAN8

References
1. Alberts, Bruce (2002). "Chapter 7.6". Molecular biology of the cell. Alexander
Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter (4 ed.). New
York: Garland Science. ISBN 0-8153-3218-1. OCLC 48122761.
2. New England BioLabs. "T7 Expression". Accessed Oct 4 2021. Accessible.
3. Shilling, Patrick J.; Mirzadeh, Kiavash; Cumming, Alister J.; Widesheim,
Magnus; Köck, Zoe; Daley, Daniel O. (2020-05-07). "Improved designs for
pET expression plasmids increase protein production yield in Escherichia
coli". Communications Biology. 3 (1): 214. doi:10.1038/s42003-020-0939-
8. ISSN 2399-3642. PMC 7205610. PMID 32382055.
4. Jump up to:a b Karen McNulty Walsh. "The Science Behind the Shot: Biotech
Tools Developed at Brookhaven Lab Fundamental to Making COVID-19
Vaccines." Brookhaven National Laboratory. April 13, 2021. Accessed Oct 4
2021.
5. Diane Greenberg. "F. William Studier: Basic Research Leads to Most
Successful BNL Technology." Brookhaven National Laboratory. April 7, 2011.
Accessed Oct 4 2021. Accessible.
6. Tyasning Kroemer. "How Does IPTG Induction Work?." GOLDBIO.
Accessed Oct 4 2021. Accessible.
7. Iost, I; Guillerez, J; Dreyfus, M (1992). "Bacteriophage T7 RNA polymerase
travels far ahead of ribosomes in vivo". Journal of Bacteriology. 174 (2): 619–
622. doi:10.1128/jb.174.2.619-622.1992. ISSN 0021-
9193. PMC 205757. PMID 1729251.
8. Stano, Natalie M.; Patel, Smita S. (2004-04-16). "T7 Lysozyme Represses T7
RNA Polymerase Transcription by Destabilizing the Open Complex during
Initiation *". Journal of Biological Chemistry. 279 (16): 16136–
16143. doi:10.1074/jbc.M400139200. ISSN 0021-9258. PMID 14764584.
9. Carl MacGowan. "Accidental BNL find now key building block for two
COVID-19 vaccines." NewsDay. May 24 2021. Accessed Oct 4 2021.