Plasmid 101: Protein Expression (2023)

The central principle of molecular biology isADN→ARN→Proteína. To synthesize a specific protein, the DNA must first be transcribed into messenger RNA (mRNA). The mRNA can then be translated in the ribosome into polypeptide chains that form the primary structure of proteins. Then, most proteins are modified through a series of post-translational modifications, including protein folding, disulfide bond formation, glycosylation, and acetylation, to create stable and functional proteins. Protein expression refers to the second step in this process: the synthesis of proteins from the mRNA and the addition of post-translational modifications.

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Researchers use different techniques to control protein expression for experimental, biotechnological, and medical applications. Research can make proteins visiblelivemark withfluorescent proteinsto study the localization or purify proteins to study their structure, interactions and functions. Proteins can also be purified for use in molecular biology research (eg, polymerases and other enzymes can be purified and used to manipulate DNA) or in medicine (eg, insulin).

Proteins must be made using complex cell-based concoctions or using living cells, unlike DNA, which can be synthesized relatively easily. There are several types of expression systems used for the production and purification of proteins. These includeMammalian, insect, bacterial, plant, yeast and cell-free expression systems.

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Plasmid 101: Protein Expression (1)

In general, the general strategy for protein expression is to transfect cells with the DNA template of your choice and allow those cells to transcribe, translate, and modify the desired protein. The modified proteins can then be extracted using lysed cell proteins.Signand separated from contaminants using a variety of purification processes. The decision of which expression system to use depends on several factors:

  1. The protein you want to express
  2. How much protein do you need?
  3. Your plans for subsequent applications

In this blog post, we outline some of the most common expression systems, including their benefits and caveats to consider before settling on a system.

Mammalian expression systems

Mammalian cells are an ideal system for expressing mammalian proteins that require multiple post-translational modifications for proper protein function. Most DNA constructs designed for mammalian expression use viral promoters (SV40, CMV and RSV) for robust expression after transfection. Mammalian systems can express proteins transiently or through stable cell lines. If the transfection is successful, both methods lead to high protein yields.

Some mammalian systems also allow control of when a protein is expressed through the use of housekeeping proteins andinducible promoters. Inducible promoters are extremely useful when a desired protein product is toxic to cells at high concentrations. Despite their advantages, mammalian expression systems require demanding cell culture conditions compared to other systems.

insect expression systems

Insect cells can also be used to make complex eukaryotic proteins with the appropriate post-translational modifications. There are two types of insect expression systems; baculovirus-infected non-lytic insect cells.

(Video) Expression vectors | What is in an expression vector? | applications of expression vectors

Baculovirus expression systems are very powerful for high level expression of recombinant proteins. These systems allow high expression of very complex glycosylated proteins that cannot be produced inE. colior yeast cells. The only problem with baculovirus systems is that the infected host cell eventually lyses. Cell lysis stops protein production, but there are non-lytic insect cell expression systems (sf9, Sf21, Hi-5 cells) that allow continuous expression of genes integrated into the insect cell genome.Both types of insect expression systems can be scaled up to produce large amounts of protein.

Some disadvantages of insect cell expression systems include that virus production can take a long time and that insect cells require demanding culture conditions similar to those of mammalian expression systems.

bacterialexpression systems

When someone wants to produce large amounts of protein quickly and cheaply, a bacterial host cell is almost always the answer.E. coliis definitely one of the most popular hosts for the expression of proteins with differentdeformationspecialized in protein expression. Protein expression in bacteria is quite simple; The DNA encoding your protein of interest is inserted into a plasmid expression vector, which is thentransformed into bacterial cells. The transformed cells proliferate, are induced to produce their protein of interest, and are then lysed. The protein can then be purified from cell debris.

There are several popular DNA vectors that can be used to produce large amounts of protein in bacterial cells: for example, the pET, pRSET, Gateway pDEST, and pBAD vectors. Protein expression from each of these vectors is controlled by a different promoter, resulting in different levels of expression for each vector; Lower expression may be required if your protein is toxicE. coli.Of all the vectors, pET produces the highest level of protein expression under the control of the T7 lac promoter and induced by lactose.

Despite their ease of manipulation, it is important to note that bacteria are generally unable to produce functional multi-domain mammalian proteins because bacterial cells are not equipped to add appropriate post-translational modifications. Furthermore, many proteins produced by bacteria become insoluble, forming inclusion bodies that are difficult to extract without patience and hard reagents.

Plantarexpression systems

Plants offer an inexpensive, low-tech means for the massive expression of recombinant proteins. Many cells from different plant species such as maize, tobacco, rice, sugar cane and even potato tubers have been used for protein expression.

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Plant systems share many of the same features and processing requirements as mammalian cell expression systems, including more complex post-translational modifications. However, the extraction and purification of recombinant plant proteins can be expensive and time consuming because plant tissues themselves are biochemically complex.

To get around these problems, the scientists took advantage of the natural secretion of biochemicals and proteins from plant roots. Labeling of recombinant proteins with a naturally secreted plant peptide allows for easier access and purification of a desired protein. Although this is a very young technology, plant cells have been used to express a wide range of proteins, including antibodies and pharmaceuticals, particularly interleukins.

Yeast Expression Systems

Yeast is an excellent expression system for producing large amounts of recombinant eukaryotic proteins. Although many yeast species can be used for protein expression,S. cerevisiaeit is the most reliable and commonly used species due to its use as a model organism in genetics and biochemistry.

when you useS. cerevisiaeinvestigators generally place recombinant proteins under the control of the galactose-inducible (GAL) promoter. Other commonly used promoters include the phosphate and copper inducible PHO5 and CUP1 promoters, respectively. Yeast cells are grown in well-defined media and can be easily adapted for fermentation, allowing stable large-scale protein production.

In general, yeast expression systems are easier and cheaper to manipulate than mammalian cells and, unlike bacterial systems, can often modify complex proteins. However, yeast cells have a slower growth rate than bacterial cells, and growth conditions often need to be optimized. Yeast cells are also known to hyperglycosylate proteins, which can be a problem depending on the protein chosen.

Cell Free Expression Systems

In cell-free expression systems, proteins are assembledin vitrousing purified components of the transcription and translation machinery. These include ribosomes, RNA polymerase, tRNA, ribonucleotides, and amino acids. Cell-free expression systems are ideal for rapid assembly of more than one protein in a reaction. A key benefit of this system of systems is its ability to assemble proteins with tagged or modified amino acids useful in various downstream applications. However, cell-free expression systems are expensive and technically demanding to use.

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Plasmid 101: Protein Expression (2)Alyssa D. Cecchetelli is a scientist at Addgene. She received her Ph.D. from Northeastern University and has a special interest in cell signaling and communication. She loves being able to help the scientific community share plasmids.

additional resources

Resources on the Addgene blog

  • Learninducible promoters
  • Learnoppressive prosecutors
  • Boxprotein labels

Resources at

  • Bacterial Expression Systems
  • Search plasmids by expression system

Plasmid 101: Protein Expression (3)

Subjects:plasmid 101,plasmid

(Video) Plasmid design (bacterial expression vector)

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1. The Promoter Region - Plasmids 101
2. Origin of Replication - Plasmids 101
3. Generation of Plasmids Expressing FLAG-tagged Proteins with Human Elongation Factor-1α Promoter
(New England Biolabs)
4. Expression Plasmids for Every Gene - Ready-to-Transfect
(OriGene Technologies Inc.)
5. Plasmid DNA Transfection Protocol
(Thermo Fisher Scientific)
6. Expression Systems
(UGA Master of Biomanufacturing and Bioprocessing)
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