T7 expression system

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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]

Contents

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 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]

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]

Related Research Articles

<span class="mw-page-title-main">Lambda phage</span> Bacteriophage that infects Escherichia coli

Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

<span class="mw-page-title-main">Promoter (genetics)</span> Region of DNA encouraging transcription

In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA . Promoters can be about 100–1000 base pairs long, the sequence of which is highly dependent on the gene and product of transcription, type or class of RNA polymerase recruited to the site, and species of organism.

<span class="mw-page-title-main">Protein production</span>

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

A transcriptional activator is a protein that increases transcription of a gene or set of genes. Activators are considered to have positive control over gene expression, as they function to promote gene transcription and, in some cases, are required for the transcription of genes to occur. Most activators are DNA-binding proteins that bind to enhancers or promoter-proximal elements. The DNA site bound by the activator is referred to as an "activator-binding site". The part of the activator that makes protein–protein interactions with the general transcription machinery is referred to as an "activating region" or "activation domain".

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

<span class="mw-page-title-main">Repressor</span> Sort of RNA-binding protein in molecular genetics

In molecular genetics, a repressor is a DNA- or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking or reducing of expression is called repression.

<span class="mw-page-title-main">Silencer (genetics)</span> Type of DNA sequence

In genetics, a silencer is a DNA sequence capable of binding transcription regulation factors, called repressors. DNA contains genes and provides the template to produce messenger RNA (mRNA). That mRNA is then translated into proteins. When a repressor protein binds to the silencer region of DNA, RNA polymerase is prevented from transcribing the DNA sequence into RNA. With transcription blocked, the translation of RNA into proteins is impossible. Thus, silencers prevent genes from being expressed as proteins.

In eukaryote cells, RNA polymerase III is a protein that transcribes DNA to synthesize 5S ribosomal RNA, tRNA, and other small RNAs.

<span class="mw-page-title-main">Termination signal</span>

A termination signal is a sequence that signals the end of transcription or translation. Termination signals are found at the end of the part of the chromosome being transcribed during transcription of mRNA. Termination signals bring a stop to transcription, ensuring that only gene-encoding parts of the chromosome are transcribed. Transcription begins at the promoter when RNA polymerase, an enzyme that facilitates transcription of DNA into mRNA, binds to a promoter, unwinds the helical structure of the DNA, and uses the single-stranded DNA as a template to synthesize RNA. Once RNA polymerase reaches the termination signal, transcription is terminated. In bacteria, there are two main types of termination signals: intrinsic and factor-dependent terminators. In the context of translation, a termination signal is the stop codon on the mRNA that elicits the release of the growing peptide from the ribosome.

<span class="mw-page-title-main">T7 RNA polymerase</span> Class of enzymes

T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'→ 3' direction.

<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. Repression of gene expression for this operon works via binding of repressor molecules to two operators. These repressors dimerize, creating a loop in the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus prevent transcription. Additionally, since the metabolism of galactose in the cell is involved in both anabolic and catabolic pathways, a novel regulatory system using two promoters for differential repression has been identified and characterized within the context of the gal operon.

The lacUV5 promoter is a mutated promoter from the Escherichia coli lac operon which is used in molecular biology to drive gene expression on a plasmid. lacUV5 is very similar to the classical lac promoter, containing just 2 base pair mutations in the -10 hexamer region, compared to the lac promoter. LacUV5 is among the most commonly used promoters in molecular biology because it requires no additional activators and it drives high levels of gene expression.

<span class="mw-page-title-main">Tac-Promoter</span>

The Tac-Promoter, or tac vector is a synthetically produced DNA promoter, produced from the combination of promoters from the trp and lac operons. It is commonly used for protein production in Escherichia coli.

Charles Clifton Richardson is an American biochemist and professor at Harvard University. Richardson received his undergraduate education at Duke University, where he majored in medicine. He received his M.D. at Duke Medical School in 1960. Richardson works as a professor at Harvard Medical School, and he served as editor/associate editor of the Annual Review of Biochemistry from 1972 to 2003. Richardson received the American Chemical Society Award in Biological Chemistry in 1968, as well as numerous other accolades.

Phage-assisted continuous evolution (PACE) is a phage-based technique for the automated directed evolution of proteins. It relies on relating the desired activity of a target protein with the fitness of an infectious bacteriophage which carries the protein's corresponding gene. Proteins with greater desired activity hence confer greater infectivity to their carrier phage. More infectious phage propagate more effectively, selecting for advantageous mutations. Genetic variation is generated using error-prone polymerases on the phage vectors, and over time the protein accumulates beneficial mutations. This technique is notable for performing hundreds of rounds of selection with minimal human intervention.

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. 1 2 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.
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