Protein expression
Encyclopedia
Protein expression is a subcomponent of gene expression
. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins. Protein expression is commonly used by proteomics
researchers to denote the measurement of the presence and abundance of one or more proteins in a particular cell or tissue.
Protein expression systems are very widely used in the life sciences, biotechnology
and medicine
. Molecular biology
research uses an enormous number of proteins and enzymes many of which are from expression systems; particularly DNA polymerase
for PCR, reverse transcriptase
for RNA analysis and restriction endonucleases for cloning. There are also significant medical applications for expression systems, notably the production of human insulin
to treat diabetes.
, yeast
, baculovirus
/insect
, and mammalian cells.
, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level". Expression is often done to a very high level and therefore referred to as overexpression.
There are many ways to introduce foreign DNA to a cell for expression, and there are many different host cells which may be used for expression - each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the host
and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria
(such as E.coli
, B. subtilis), yeast
(such as S.cerevisiae
) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are virus
es (such as baculovirus
, retrovirus
, adenovirus), plasmid
s, artificial chromosome
s and bacteriophage
(such as lambda
). The best expression system of choice depends on the gene involved, for example the Saccharomyces cerevisiae
is often preferred for proteins that require significant posttranslational modification
and Insect
or mammal
cell lines are used when human-like splicing of the mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography
or nuclear magnetic resonance
experiments for structure determination.
E. coli is one of the most widely used expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E. coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting trancription.
For example a DNA sequence for a protein of interest could be cloned or subcloned
into a high copy-number plasmid
containing the lac
promoter, which is then transformed
into the bacterium Escherichia coli
. Addition of IPTG
(a lactose
analog) activates the lac promoter and causes the bacteria to express the protein of interest.
are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine
, components of human food, animal feed, and pharmaceutical products.
Expression of functionally active human epidermal growth factor
has been done in C. glutamicum, thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general secretory pathway
(Sec) or the twin-arginine translocation pathway (Tat).
Unlike gram-negative bacteria, the gram-positive Corynebacterium lack lipopolysaccarides that function as antigenic endotoxins in humans.
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins. Protein expression is commonly used by proteomics
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
researchers to denote the measurement of the presence and abundance of one or more proteins in a particular cell or tissue.
Protein expression systems are very widely used in the life sciences, biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
. Molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
research uses an enormous number of proteins and enzymes many of which are from expression systems; particularly DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
for PCR, reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
for RNA analysis and restriction endonucleases for cloning. There are also significant medical applications for expression systems, notably the production of human insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
to treat diabetes.
Expression systems
Commonly used protein expression systems include those derived from bacteriaBacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, baculovirus
Baculovirus
The baculoviruses are a family of large rod-shaped viruses that can be divided to two genera: nucleopolyhedroviruses and granuloviruses . While GVs contain only one nucleocapsid per envelope, NPVs contain either single or multiple nucleocapsids per envelope. The enveloped virions are further...
/insect
Insect
Insects are a class of living creatures within the arthropods that have a chitinous exoskeleton, a three-part body , three pairs of jointed legs, compound eyes, and two antennae...
, and mammalian cells.
Cell-based systems
The oldest and most widely used expression systems are cell-based and may be defined as the "combination of an expression vectorExpression vector
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level". Expression is often done to a very high level and therefore referred to as overexpression.
There are many ways to introduce foreign DNA to a cell for expression, and there are many different host cells which may be used for expression - each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the host
Host (biology)
In biology, a host is an organism that harbors a parasite, or a mutual or commensal symbiont, typically providing nourishment and shelter. In botany, a host plant is one that supplies food resources and substrate for certain insects or other fauna...
and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
(such as E.coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, B. subtilis), yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
(such as S.cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
) or eukaryotic cell lines. Common DNA sources and delivery mechanisms are virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
es (such as baculovirus
Baculovirus
The baculoviruses are a family of large rod-shaped viruses that can be divided to two genera: nucleopolyhedroviruses and granuloviruses . While GVs contain only one nucleocapsid per envelope, NPVs contain either single or multiple nucleocapsids per envelope. The enveloped virions are further...
, retrovirus
Retrovirus
A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
, adenovirus), plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s, artificial chromosome
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...
s and bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
(such as lambda
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
). The best expression system of choice depends on the gene involved, for example the Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
is often preferred for proteins that require significant posttranslational modification
Posttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....
and Insect
Insect
Insects are a class of living creatures within the arthropods that have a chitinous exoskeleton, a three-part body , three pairs of jointed legs, compound eyes, and two antennae...
or mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
cell lines are used when human-like splicing of the mRNA is required. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
or nuclear magnetic resonance
Nuclear magnetic resonance
Nuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
experiments for structure determination.
Escherichia coli
For example a DNA sequence for a protein of interest could be cloned or subcloned
Subcloning
In molecular biology, subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality....
into a high copy-number plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
containing the lac
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
promoter, which is then transformed
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
into the bacterium Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
. Addition of IPTG
IPTG
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology reagent.This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon...
(a lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
analog) activates the lac promoter and causes the bacteria to express the protein of interest.
Corynebacterium
Non-pathogenic species of the gram-positive CorynebacteriumCorynebacterium
Corynebacterium is a genus of Gram-positive rod-shaped bacteria. They are widely distributed in nature and are mostly innocuous. Some are useful in industrial settings such as C. glutamicum. Others can cause human disease. C...
are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine
Lysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
, components of human food, animal feed, and pharmaceutical products.
Expression of functionally active human epidermal growth factor
Epidermal growth factor
Epidermal growth factor or EGF is a growth factor that plays an important role in the regulation of cell growth, proliferation, and differentiation by binding to its receptor EGFR...
has been done in C. glutamicum, thus demonstrating a potential for industrial-scale production of human proteins. Expressed proteins can be targeted for secretion through either the general secretory pathway
Secretory pathway
The secretory pathway is a series of steps a cell uses to move proteins out of the cell; a process known as secretion. The path of a protein destined for secretion has its origins in the rough endoplasmic reticulum, a membrane-bound compartment in the cell...
(Sec) or the twin-arginine translocation pathway (Tat).
Unlike gram-negative bacteria, the gram-positive Corynebacterium lack lipopolysaccarides that function as antigenic endotoxins in humans.