![Genetic code](https://upload.wikimedia.org/wikipedia/commons/3/37/Genetic_code.svg)
Protein production is the
biotechnological
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...
process of generating a specific
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
. It is typically achieved by the manipulation of
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
in an organism such that it
expresses
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, ...
large amounts of a
recombinant gene
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fou ...
. This includes the
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
of the
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
to messenger
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
(
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
), the
translation
Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
of mRNA into
polypeptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A p ...
chains, which are ultimately folded into functional
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
and may be
targeted to specific subcellular or extracellular locations.
Protein production systems (also known as
expression systems) are used in the
life sciences
This list of life sciences comprises the branches of science that involve the scientific study of life – such as microorganisms, plants, and animals including human beings. This science is one of the two major branches of natural science, the ...
,
biotechnology
Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used b ...
, and
medicine
Medicine is the science and practice of caring for a patient, managing the diagnosis, prognosis, prevention, treatment, palliation of their injury or disease, and promoting their health. Medicine encompasses a variety of health care pract ...
.
Molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
research uses numerous proteins and enzymes, many of which are from expression systems; particularly
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...
for
PCR,
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
for RNA analysis,
restriction endonuclease
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s for cloning, and to make proteins that are screened in
drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which new candidate medications are discovered.
Historically, drugs were discovered by identifying the active ingredient from traditional remedies or by ...
as
biological target
A biological target is anything within a living organism to which some other entity (like an endogenous ligand or a drug) is directed and/or binds, resulting in a change in its behavior or function. Examples of common classes of biological targets ...
s or as potential drugs themselves. There are also significant applications for expression systems in
industrial fermentation
Industrial fermentation is the intentional use of fermentation in manufacturing products useful to humans. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical industry ...
, notably the production of
biopharmaceuticals
A biopharmaceutical, also known as a biological medical product, or biologic, is any pharmaceutical drug product manufactured in, extracted from, or semisynthesized from biological sources. Different from totally synthesized pharmaceuticals, t ...
such as human
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
to treat
diabetes
Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ap ...
, and to manufacture
enzymes
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
.
Protein production systems
Commonly used protein production systems include those derived from
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
,
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
,
baculovirus
''Baculoviridae'' is a family of viruses. Arthropod
Arthropods (, (gen. ποδός)) are invertebrate animals with an exoskeleton, a Segmentation (biology), segmented body, and paired jointed appendages. Arthropods form the phylum Arthropod ...
/
insect
Insects (from Latin ') are pancrustacean hexapod invertebrates of the class Insecta. They are the largest group within the arthropod phylum. Insects have a chitinous exoskeleton, a three-part body ( head, thorax and abdomen), three pairs ...
,
mammalian
Mammals () are a group of vertebrate animals constituting the class (biology), class Mammalia (), characterized by the presence of mammary glands which in Female#Mammalian female, females produce milk for feeding (nursing) their young, a ...
cells,
and more recently filamentous fungi such as ''
Myceliophthora thermophila''.
When biopharmaceuticals are produced with one of these systems, process-related impurities termed
host cell protein
Host cell proteins (HCPs) are process-related protein impurities that are produced by the host organism during biotherapeutic manufacturing and production. During the purification process, a majority of produced HCPs are removed from the final pro ...
s also arrive in the final product in trace amounts.
Cell-based systems
The oldest and most widely used expression systems are cell-based and may be defined as the "''combination of an
expression vector
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 ...
, 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''".
Overexpression is an abnormally and excessively high level of
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
which produces a pronounced gene-related
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
.
There are many ways to introduce foreign
DNA to a cell for expression, and many different host cells may be used for expression — each expression system has distinct advantages and liabilities. Expression systems are normally referred to by the
host
A host is a person responsible for guests at an event or for providing hospitality during it.
Host may also refer to:
Places
* Host, Pennsylvania, a village in Berks County
People
*Jim Host (born 1937), American businessman
* Michel Host ...
and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
(such as ''
E.coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Esche ...
'', ''
B. subtilis
''Bacillus subtilis'', known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacillu ...
''),
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
(such as ''
S.cerevisiae
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been ...
''
) or eukaryotic
cell lines
An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
. Common DNA sources and delivery mechanisms are
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
es (such as
baculovirus
''Baculoviridae'' is a family of viruses. Arthropod
Arthropods (, (gen. ποδός)) are invertebrate animals with an exoskeleton, a Segmentation (biology), segmented body, and paired jointed appendages. Arthropods form the phylum Arthropod ...
,
retrovirus
A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...
,
adenovirus
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
),
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s,
artificial chromosome Artificial chromosome may refer to:
* Yeast artificial chromosome
* Bacterial artificial chromosome
* Human artificial chromosome A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human ce ...
s and
bacteriophage
A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...
(such as
lambda
Lambda (}, ''lám(b)da'') is the 11th letter of the Greek alphabet, representing the voiced alveolar lateral approximant . In the system of Greek numerals, lambda has a value of 30. Lambda is derived from the Phoenician Lamed . Lambda gave rise ...
). The best expression system depends on the
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
involved, for example the ''
Saccharomyces cerevisiae
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...
'' is often preferred for proteins that require significant
posttranslational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
.
Insect
Insects (from Latin ') are pancrustacean hexapod invertebrates of the class Insecta. They are the largest group within the arthropod phylum. Insects have a chitinous exoskeleton, a three-part body ( head, thorax and abdomen), three pairs ...
or
mammal
Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur or ...
cell lines are used when human-like splicing of 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 is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
or
nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
experiments for structure determination.
Because bacteria are
prokaryote
A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Connec ...
s, they are not equipped with the full enzymatic machinery to accomplish the required post-translational modifications or molecular folding. Hence, multi-domain eukaryotic proteins expressed in bacteria often are non-functional. Also, many proteins become insoluble as inclusion bodies that are difficult to recover without harsh denaturants and subsequent cumbersome protein-refolding.
To address these concerns, expressions systems using multiple eukaryotic cells were developed for applications requiring the proteins be conformed as in, or closer to eukaryotic organisms: cells of plants (i.e. tobacco), of insects or mammalians (i.e. bovines) are transfected with genes and cultured in suspension and even as tissues or whole organisms, to produce fully folded proteins. Mammalian ''
in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' expression systems have however low yield and other limitations (time-consuming, toxicity to host cells,..). To combine the high yield/productivity and scalable protein features of bacteria and yeast, and advanced epigenetic features of plants, insects and mammalians systems, other protein production systems are developed using unicellular eukaryotes (i.e. non-pathogenic '
''
Leishmania
''Leishmania'' is a parasitic protozoan, a single-celled organism of the genus '' Leishmania'' that are responsible for the disease leishmaniasis. They are spread by sandflies of the genus ''Phlebotomus'' in the Old World, and of the genus '' ...
''
' cells).
Bacterial systems
= ''Escherichia coli''
=
![EscherichiaColi NIAID](https://upload.wikimedia.org/wikipedia/commons/3/32/EscherichiaColi_NIAID.jpg)
''
E. coli'' is one of the most widely used expression hosts, and DNA is normally introduced in a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
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 transcription.
For example, a DNA sequence for a protein of interest could be
cloned
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, c ...
or
subcloned into a high copy-number plasmid containing the ''
lac
Lac is the resinous secretion of a number of species of lac insects, of which the most commonly cultivated is ''Kerria lacca''.
Cultivation begins when a farmer gets a stick that contains eggs ready to hatch and ties it to the tree to be infes ...
'' (often
LacUV5
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 ...
) promoter, which is then
transformed into the bacterium ''E. coli''. Addition of
IPTG (a
lactose
Lactose is a disaccharide sugar synthesized by galactose and glucose subunits and has the molecular formula C12H22O11. Lactose makes up around 2–8% of milk (by mass). The name comes from ' (gen. '), the Latin word for milk, plus the suffix '' - ...
analog) activates the lac promoter and causes the bacteria to express the protein of interest.
''E. coli'' strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack ''
lon'' and ''
OmpT
OmpT is an aspartyl protease found on the outer membrane of ''Escherichia coli''. OmpT is a subtype of the family of omptin proteases, which are found on some gram-negative species of bacteria.
Structure
OmpT is a 33.5 kDa outer membrane protei ...
'' proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides
T7 RNA polymerase
T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'→ 3' direction.
Activity
T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promo ...
(driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead.
= ''Corynebacterium''
=
Non-pathogenic species of the gram-positive ''
Corynebacterium'' are used for the commercial production of various amino acids. The ''
C. glutamicum'' species is widely used for producing
glutamate
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
and
lysine
Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −C ...
, components of human food, animal feed and pharmaceutical products.
Expression of functionally active human
epidermal growth factor
Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. Human EGF is 6-k Da and has 53 amino acid residues and three intramolecular disulfide bonds.
EGF was originally descr ...
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 440px
Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classical ...
(Sec) or the
twin-arginine translocation pathway
The twin-arginine translocation pathway (Tat pathway) is a protein export, or secretion pathway found in plants, bacteria, and archaea. In contrast to the Sec pathway which transports proteins in an unfolded manner, the Tat pathway serves to ac ...
(Tat).
Unlike
gram-negative bacteria
Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
, the gram-positive ''Corynebacterium'' lack
lipopolysaccharides
Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a polysaccharide that are bacterial toxins. They are composed of an O-antigen, an outer core, and an inner core all joined by a covalent bond, and are found in the outer me ...
that function as antigenic
endotoxins
Lipopolysaccharides (LPS) are large molecules consisting of a lipid and a polysaccharide that are bacterial toxins. They are composed of an O-antigen, an outer core, and an inner core all joined by a covalent bond, and are found in the outer me ...
in humans.
= ''Pseudomonas fluorescens''
=
The non-pathogenic and gram-negative bacteria, ''
Pseudomonas fluorescens
''Pseudomonas fluorescens'' is a common Gram-negative, rod-shaped bacterium. It belongs to the ''Pseudomonas'' genus; 16S rRNA analysis as well as phylogenomic analysis has placed ''P. fluorescens'' in the ''P. fluorescens'' group within the genu ...
'', is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. '' P. fluorescens'' is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. ''P. fluorescens'' is most well known for its ability to rapid and successfully produce high titers of active, soluble protein.
Eukaryotic systems
= Yeasts
=
Expression systems using either ''
S. cerevisiae
''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have bee ...
'' or ''
Pichia pastoris
''Pichia pastoris'' is a species of methylotrophic yeast. It was found in the 1960s, with its feature of using methanol as a source of carbon and energy. After years of study, ''P. pastoris'' was widely used in biochemical research and biotech ...
'' allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media of proteins.
= Filamentous fungi
=
Filamentous fungi, especially ''
Aspergillus
' () is a genus consisting of several hundred mold species found in various climates worldwide.
''Aspergillus'' was first catalogued in 1729 by the Italian priest and biologist Pier Antonio Micheli. Viewing the fungi under a microscope, Miche ...
'' and ''
Trichoderma
''Trichoderma'' is a genus of fungi in the family Hypocreaceae that is present in all soils, where they are the most prevalent culturable fungi. Many species in this genus can be characterized as opportunistic avirulent plant symbionts. This ref ...
'', but also more recently ''
Myceliophthora thermophila'' C1
have been developed into expression platforms for screening and production of diverse
industrial enzymes Industrial enzymes are enzymes that are commercially used in a variety of industries such as pharmaceuticals, chemical production, biofuels, food & beverage, and consumer products. Due to advancements in recent years, biocatalysis through isolat ...
. The expression system C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media.
= ''Baculovirus''-infected cells
=
Baculovirus
''Baculoviridae'' is a family of viruses. Arthropod
Arthropods (, (gen. ποδός)) are invertebrate animals with an exoskeleton, a Segmentation (biology), segmented body, and paired jointed appendages. Arthropods form the phylum Arthropod ...
-infected insect cells
(
Sf9
SF9 ( ko, 에스에프나인 ; shortened from Sensational Feeling 9) is a South Korean boy band formed by FNC Entertainment and the company's first dance boy group. Consisting of nine members, the group debuted on October 5, 2016 with the relea ...
,
Sf21
Sf21 (officially called IPLB-Sf21-AE) is a continuous cell line developed from ovaries of the Fall Army worm, ''Spodoptera frugiperda'', a moth species that is an agricultural pest on corn and other grass species. It was originally developed in ...
,
High Five High five is a friendly gesture in which one individual slaps another's hand.
High five (and variants such as Hi5, Hi-5, and Hi-Five) may also refer to:
Music
* Hi-5 (Australian group), an Australian children's musical group
* Hi-5 (Greek band), ...
strains) or mammalian cells (
HeLa
HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta La ...
,
HEK 293
Human embryonic kidney 293 cells, also often referred to as HEK 293, HEK-293, 293 cells, or less precisely as HEK cells, are a specific immortalised cell line derived from a spontaneously miscarried or aborted fetus or human embryonic kidney cells ...
) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.
It is useful for production of proteins in high quantity. Genes are not expressed continuously because infected host cells eventually lyse and die during each infection cycle.
= Non-lytic insect cell expression
=
Non-lytic insect cell expression is an alternative to the lytic baculovirus expression system. In non-lytic expression, vectors are transiently or stably
transfected
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to desc ...
into the chromosomal DNA of insect cells for subsequent gene expression.
This is followed by selection and screening of recombinant clones.
The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression.
Cell lines used for this system include:
Sf9
SF9 ( ko, 에스에프나인 ; shortened from Sensational Feeling 9) is a South Korean boy band formed by FNC Entertainment and the company's first dance boy group. Consisting of nine members, the group debuted on October 5, 2016 with the relea ...
,
Sf21
Sf21 (officially called IPLB-Sf21-AE) is a continuous cell line developed from ovaries of the Fall Army worm, ''Spodoptera frugiperda'', a moth species that is an agricultural pest on corn and other grass species. It was originally developed in ...
from ''
Spodoptera frugiperda
The fall armyworm (''Spodoptera frugiperda'') is a species in the order Lepidoptera and one of the species of the fall armyworm moths distinguished by their larval life stage. The term "armyworm" can refer to several species, often describing the ...
'' cells,
Hi-5 from ''
Trichoplusia ni
The cabbage looper (''Trichoplusia ni'') is a medium-sized moth in the family Noctuidae, a family commonly referred to as owlet moths. Its common name comes from its preferred host plants and distinctive crawling behavior. Cruciferous vegetab ...
'' cells, and
Schneider 2 cells Schneider 2 cells, usually abbreviated as S2 cells, are one of the most commonly used ''Drosophila melanogaster'' cell lines. S2 cells were derived from a primary culture of late stage (20–24 hours old) ''Drosophila melanogaster'' embryos by Dr. I ...
and Schneider 3 cells from ''
Drosophila melanogaster
''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...
'' cells.
With this system, cells do not lyse and several cultivation modes can be used.
Additionally, protein production runs are reproducible.
This system gives a homogeneous product.
A drawback of this system is the requirement of an additional screening step for selecting viable
clones
Clone or Clones or Cloning or Cloned or The Clone may refer to:
Places
* Clones, County Fermanagh
* Clones, County Monaghan, a town in Ireland
Biology
* Clone (B-cell), a lymphocyte clone, the massive presence of which may indicate a pathologi ...
.
= ''
Excavata
Excavata is a major supergroup of unicellular organisms belonging to the domain Eukaryota. It was first suggested by Simpson and Patterson in 1999 and introduced by Thomas Cavalier-Smith in 2002 as a formal taxon. It contains a variety of free- ...
'' =
''
Leishmania
''Leishmania'' is a parasitic protozoan, a single-celled organism of the genus '' Leishmania'' that are responsible for the disease leishmaniasis. They are spread by sandflies of the genus ''Phlebotomus'' in the Old World, and of the genus '' ...
tarentolae'' (cannot infect mammals) expression systems allow stable and lasting production of proteins at high yield, in chemically defined media. Produced proteins exhibit fully eukaryotic post-translational modifications, including
glycosylation
Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not al ...
and disulfide bond formation.
= Mammalian systems
=
The most common mammalian expression systems are
Chinese Hamster ovary
The ovary is an organ in the female reproductive system that produces an ovum. When released, this travels down the fallopian tube into the uterus, where it may become fertilized by a sperm. There is an ovary () found on each side of the body. ...
(CHO) and Human embryonic kidney (HEK) cells.
*
Chinese hamster ovary cell
Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have foun ...
*
Mouse
A mouse ( : mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
myeloma
lymphoblstoid (e.g. NS0 cell)
* Fully Human
** Human embryonic kidney cells (
HEK-293)
** Human embryonic retinal cells (Crucell's Per.C6)
** Human
amniocyte cells (Glycotope and CEVEC)
Cell-free systems
Cell-free production of proteins is performed ''in vitro'' using purified RNA polymerase, ribosomes, tRNA and ribonucleotides. These reagents may be produced by extraction from cells or from a cell-based expression system. Due to the low expression levels and high cost of cell-free systems, cell-based systems are more widely used.
See also
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Cellosaurus
Cellosaurus is an online knowledge base on cell lines, which attempts to document all cell lines used in biomedical research. It is provided by the Swiss Institute of Bioinformatics (SIB). It is an ELIXIR Core Data Resource as well as an IR ...
, a database of cell lines
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Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...
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Single-cell protein
Single-cell proteins (SCP) or microbial proteins refer to edible unicellular microorganisms. The biomass or protein extract from pure or mixed cultures of algae, yeasts, fungi or bacteria may be used as an ingredient or a substitute for protein-ric ...
*
Protein purification Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interact ...
*
Precision fermentation
Industrial fermentation is the intentional use of fermentation in manufacturing products useful to humans. In addition to the mass production of fermented foods and drinks, industrial fermentation has widespread applications in chemical indust ...
*
Host cell protein
Host cell proteins (HCPs) are process-related protein impurities that are produced by the host organism during biotherapeutic manufacturing and production. During the purification process, a majority of produced HCPs are removed from the final pro ...
*
List of recombinant proteins The following is a list of notable proteins that are produced from recombinant DNA, using biomolecular engineering. In many cases, recombinant human proteins have replaced the original animal-derived version used in medicine. The prefix "rh" for "r ...
References
Further reading
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External links
{{Microorganisms
Gene expression
Biotechnology