Editing Protein production

{{Short description|Biotechnological process}}
{{About|the biotechnological method|the natural process in cells|Gene expression}}

[[File:Genetic code.svg|alt=Central dogma depicting transcription from DNA code to RNA code to the proteins in the second step covering the production of protein.|thumb|[[Central dogma of biology|Central dogma]] depicting [[transcription (biology)|transcription]] from [[DNA]] code to [[RNA]] code to the [[protein]]s in the second step covering the production of protein.]]
'''Protein production''' is the [[biotechnology|biotechnological]] process of generating a specific [[protein]]. It is typically achieved by the manipulation of [[gene expression]] in an organism such that it [[heterologous expression|expresses]] large amounts of a [[recombinant gene]]. This includes the [[transcription (biology)|transcription]] of the [[recombinant DNA]] to messenger [[RNA]] ([[mRNA]]), the [[translation (biology)|translation]] of mRNA into [[polypeptide]] chains, which are ultimately folded into functional [[proteins]] and may be [[protein targeting|targeted]] to specific subcellular or extracellular locations.<ref>{{cite journal | vauthors = Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schütz A, Heinemann U, Yokoyama S, Büssow K, Gunsalus KC | display-authors = 6 | title = Protein production and purification | journal = Nature Methods | volume = 5 | issue = 2 | pages = 135–46 | date = February 2008 | pmid = 18235434 | pmc = 3178102 | doi = 10.1038/nmeth.f.202 }}</ref>

Protein production systems (also known as [[Gene expression#Expression system|expression system]]s) are used in the [[life sciences]], [[biotechnology]], and [[medicine]]. [[Molecular biology]] research uses numerous proteins and enzymes, many of which are from expression systems; particularly [[DNA polymerase]] for [[polymerase chain reaction|PCR]], [[reverse transcriptase]] for RNA analysis, [[restriction endonuclease]]s for cloning, and to make proteins that are screened in [[drug discovery]] as [[biological target]]s or as potential drugs themselves. There are also significant applications for expression systems in [[industrial fermentation]], notably the production of [[biopharmaceuticals]] such as human [[insulin]] to treat [[diabetes]], and to manufacture [[Enzyme#Industrial applications|enzymes]].

== Protein production systems ==

Commonly used protein production systems include those derived from [[bacteria]],<ref name="pmid10508629">{{cite journal | vauthors = Baneyx F | title = Recombinant protein expression in Escherichia coli | journal = Current Opinion in Biotechnology | volume = 10 | issue = 5 | pages = 411–21 | date = October 1999 | pmid = 10508629 | doi = 10.1016/s0958-1669(99)00003-8 }}</ref><ref name=":1">{{Cite journal |last1=Rosano |first1=Germán |last2=Ceccarelli |first2=Eduardo |date=2014-04-17 |title=Recombinant protein expression in Escherichia coli: advances and challenges |journal=Frontiers in Microbiology |volume=5 |page=172 |doi=10.3389/fmicb.2014.00172 |pmid=24860555 |pmc=4029002 |doi-access=free }}</ref> [[yeast]],<ref name="pmid11098467">{{cite journal | vauthors = Cregg JM, Cereghino JL, Shi J, Higgins DR | title = Recombinant protein expression in Pichia pastoris | journal = Molecular Biotechnology | volume = 16 | issue = 1 | pages = 23–52 | date = September 2000 | pmid = 11098467 | doi = 10.1385/MB:16:1:23 | s2cid = 35874864 | doi-access = free }}</ref><ref name="pmid21943899">{{cite book | vauthors = Malys N, Wishart JA, Oliver SG, McCarthy JE | title = Methods in Systems Biology | chapter = Protein production in Saccharomyces cerevisiae for systems biology studies | series = Methods in Enzymology | volume = 500 | pages = 197–212 | year = 2011 | pmid = 21943899 | doi = 10.1016/B978-0-12-385118-5.00011-6 | isbn = 9780123851185 }}</ref> [[baculovirus]]/[[insect]],<ref name="pmid15877075">{{cite journal | vauthors = Kost TA, Condreay JP, Jarvis DL | title = Baculovirus as versatile vectors for protein expression in insect and mammalian cells | journal = Nature Biotechnology | volume = 23 | issue = 5 | pages = 567–75 | date = May 2005 | pmid = 15877075 | pmc = 3610534 | doi = 10.1038/nbt1095 }}</ref> [[mammalian]] cells,<ref name="pmid15766864">{{cite journal | vauthors = Rosser MP, Xia W, Hartsell S, McCaman M, Zhu Y, Wang S, Harvey S, Bringmann P, Cobb RR | title = Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system | journal = Protein Expression and Purification | volume = 40 | issue = 2 | pages = 237–43 | date = April 2005 | pmid = 15766864 | doi = 10.1016/j.pep.2004.07.015 }}</ref><ref name="pmid18541133">{{cite journal | vauthors = Lackner A, Genta K, Koppensteiner H, Herbacek I, Holzmann K, Spiegl-Kreinecker S, Berger W, Grusch M | title = A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells | journal = Analytical Biochemistry | volume = 380 | issue = 1 | pages = 146–8 | date = September 2008 | pmid = 18541133 | doi = 10.1016/j.ab.2008.05.020 }}</ref> and more recently filamentous fungi such as ''[[Myceliophthora thermophila]]''.<ref name="Visser 214–223">{{Cite journal |last1=Visser |first1=Hans |last2=Joosten |first2=Vivi |last3=Punt |first3=Peter J. |last4=Gusakov |first4=Alexander V. |last5=Olson |first5=Phil T. |last6=Joosten |first6=Rob |last7=Bartels |first7=Jeffrey |last8=Visser |first8=Jaap |last9=Sinitsyn |first9=Arkady P. |display-authors=6 |name-list-style=vanc |date=June 2011 |title=Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1 |journal=Industrial Biotechnology |volume=7 |issue=3 |pages=214–223 |doi=10.1089/ind.2011.7.214 |quote=Aspergillus and Trichoderma are currently the main fungal genera used to produce industrial enzymes.}}</ref> When biopharmaceuticals are produced with one of these systems, process-related impurities termed [[host cell protein]]s also arrive in the final product in trace amounts.<ref>{{Cite journal|last1=Wang|first1=Xing|last2=Hunter|first2=Alan K.|last3=Mozier|first3=Ned M.|date=2009-06-15|title=Host cell proteins in biologics development: Identification, quantitation and risk assessment|journal=Biotechnology and Bioengineering|language=en|volume=103|issue=3|pages=446–458|doi=10.1002/bit.22304|pmid=19388135|s2cid=22707536|issn=0006-3592|doi-access=free}}</ref>

===Cell-based systems===
{{more footnotes|date=January 2024}}
The oldest and most widely used expression systems are cell-based and may be defined as the "''combination of an [[expression vector]], 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''".<ref name="urlDefinition: expression system from Online Medical Dictionary">{{cite web | url = http://cancerweb.ncl.ac.uk/cgi-bin/omd?expression+system | title = Definition: expression system  | date = 1997-11-13 | work = Online Medical Dictionary | publisher = Centre for Cancer Education, University of Newcastle upon Tyne: Cancerweb | access-date = 2008-06-10}}</ref><ref name="urlExpression system">{{cite web | url = http://www.biology-online.org/dictionary/Expression_system | title = Expression system - definition | date = 2005-10-03 | work = Biology Online | publisher = Biology-Online.org | access-date = 2008-06-10}}</ref> Overexpression is an abnormally and excessively high level of [[gene expression]] which produces a pronounced gene-related [[phenotype]].<ref name="Overexpression - Oxford">{{cite web|title=overexpression|url=https://en.oxforddictionaries.com/definition/overexpression|archive-url=https://web.archive.org/web/20180210181025/https://en.oxforddictionaries.com/definition/overexpression|url-status=dead|archive-date=February 10, 2018|website=Oxford Living Dictionary|publisher=Oxford University Press|access-date=18 May 2017|date=2017|quote=The production of abnormally large amounts of a substance which is coded for by a particular gene or group of genes; the appearance in the phenotype to an abnormally high degree of a character or effect attributed to a particular gene.}}</ref><ref name="Overexpress - NCI">{{cite web|title=overexpress|url=https://www.cancer.gov/publications/dictionaries/cancer-terms?cdrid=45812|website=NCI Dictionary of Cancer Terms|publisher=National Cancer Institute at the National Institutes of Health|access-date=18 May 2017|quote=overexpress<br />In biology, to make too many copies of a protein or other substance. Overexpression of certain proteins or other substances may play a role in cancer development.|date=2011-02-02}}</ref>{{clarify|reason=Why bring it up? (to make more of the desired protein)|date=January 2024}}

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 (biology)|host]] and the DNA source or the delivery mechanism for the genetic material. For example, common hosts are [[bacteria]] (such as ''[[Escherichia coli|E. coli]]'', ''[[B. subtilis]]''), [[yeast]] (such as ''[[Saccharomyces cerevisiae|S. cerevisiae]]''<ref name="pmid21943899"/>) or eukaryotic [[cell lines]]. Common DNA sources and delivery mechanisms are [[virus]]es (such as [[baculovirus]], [[retrovirus]], [[adenovirus]]), [[plasmid]]s, [[Bacterial artificial chromosome|artificial chromosome]]s and [[bacteriophage]] (such as [[lambda phage|lambda]]). The best expression system depends on the [[gene]] involved, for example the ''[[Saccharomyces cerevisiae]]'' is often preferred for proteins that require significant [[posttranslational modification]]. [[Insect]] or [[mammal]] 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]] or [[nuclear magnetic resonance]] experiments for structure determination.

Because bacteria are [[prokaryote]]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]]'' 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 '<nowiki/>''[[Leishmania]]''<nowiki/>' cells).

==== Bacterial systems ====

===== ''Escherichia coli'' =====
[[File:E. coli Bacteria (7316101966).jpg|thumb|right|''E. coli'', one of the most popular hosts for artificial gene expression.]]
''[[Escherichia coli|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 transcription.<ref name=":1" />

For example, a DNA sequence for a protein of interest could be [[cloning#Cloning in biology|cloned]] or [[subcloning|subcloned]] into a high copy-number plasmid containing the ''[[lac operon|lac]]'' (often [[LacUV5]]) promoter, which is then [[transformation (genetics)|transformed]] into the bacterium ''E. coli''. Addition of [[IPTG]] (a [[lactose]] analog) activates the lac promoter and causes the bacteria to express the protein of interest.<ref name="pmid10508629" />

''E. coli'' strain BL21 and BL21(DE3) are two strains commonly used for protein production. As members of the B lineage, they lack ''[[Lon protease family|lon]]'' and ''[[OmpT]]'' proteases, protecting the produced proteins from degradation. The DE3 prophage found in BL21(DE3) provides [[T7 RNA polymerase]] (driven by the LacUV5 promoter), allowing for vectors with the T7 promoter to be used instead.<ref>{{cite journal |last1=Jeong |first1=H |last2=Barbe |first2=V |last3=Lee |first3=CH |last4=Vallenet |first4=D |last5=Yu |first5=DS |last6=Choi |first6=SH |last7=Couloux |first7=A |last8=Lee |first8=SW |last9=Yoon |first9=SH |last10=Cattolico |first10=L |last11=Hur |first11=CG |last12=Park |first12=HS |last13=Ségurens |first13=B |last14=Kim |first14=SC |last15=Oh |first15=TK |last16=Lenski |first16=RE |last17=Studier |first17=FW |last18=Daegelen |first18=P |last19=Kim |first19=JF |title=Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). |journal=Journal of Molecular Biology |date=11 December 2009 |volume=394 |issue=4 |pages=644–52 |doi=10.1016/j.jmb.2009.09.052 |pmid=19786035}}</ref>

===== ''Corynebacterium'' =====
Non-pathogenic species of the gram-positive ''[[Corynebacterium]]'' are used for the commercial production of various amino acids. The ''[[Corynebacterium glutamicum|C. glutamicum]]'' species is widely used for producing [[glutamate]] and [[lysine]],<ref>{{cite journal | vauthors = Brinkrolf K, Schröder J, Pühler A, Tauch A | title = The transcriptional regulatory repertoire of Corynebacterium glutamicum: reconstruction of the network controlling pathways involved in lysine and glutamate production | journal = Journal of Biotechnology | volume = 149 | issue = 3 | pages = 173–82 | date = September 2010 | pmid = 19963020 | doi = 10.1016/j.jbiotec.2009.12.004 }}</ref> components of human food, animal feed and pharmaceutical products.

Expression of functionally active human [[epidermal growth factor]] has been done in ''C. glutamicum'',<ref>{{cite journal | vauthors = Date M, Itaya H, Matsui H, Kikuchi Y | title = Secretion of human epidermal growth factor by Corynebacterium glutamicum | journal = Letters in Applied Microbiology | volume = 42 | issue = 1 | pages = 66–70 | date = January 2006 | pmid = 16411922 | doi = 10.1111/j.1472-765x.2005.01802.x | doi-access = free }}</ref> 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).<ref>{{cite journal | vauthors = Meissner D, Vollstedt A, van Dijl JM, Freudl R | title = Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria | journal = Applied Microbiology and Biotechnology | volume = 76 | issue = 3 | pages = 633–42 | date = September 2007 | pmid = 17453196 | doi = 10.1007/s00253-007-0934-8 | s2cid = 6238466 }}</ref>

Unlike [[gram-negative bacteria]], the gram-positive ''Corynebacterium'' lack [[lipopolysaccharides]] that function as antigenic [[endotoxins]] in humans.{{cn|date=January 2024}}

===== ''Pseudomonas fluorescens'' =====
The non-pathogenic and gram-negative bacteria, ''[[Pseudomonas fluorescens]]'', 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.<ref>{{cite journal | vauthors = Retallack DM, Jin H, Chew L | title = Reliable protein production in a Pseudomonas fluorescens expression system | journal = Protein Expression and Purification | volume = 81 | issue = 2 | pages = 157–65 | date = February 2012 | pmid = 21968453 | doi = 10.1016/j.pep.2011.09.010 }}</ref>

==== Eukaryotic systems ====

===== Yeasts =====
Expression systems using either ''[[Saccharomyces cerevisiae|S. cerevisiae]]'' or ''[[Pichia pastoris]]'' allow stable and lasting production of proteins that are processed similarly to mammalian cells, at high yield, in chemically defined media of proteins.<ref name="pmid11098467" /><ref name="pmid21943899" />

===== Filamentous fungi =====

Filamentous fungi, especially ''[[Aspergillus]]'' and ''[[Trichoderma]]'', have long been used to produce diverse [[industrial enzymes]] from their own genomes ("native", "homologous") and from recombinant DNA ("heterologous").<ref name="Visser 214–223" />

More recently, ''[[Myceliophthora thermophila]]'' C1 has been developed into an expression platform for screening and production of native and heterologous proteins.The expression system C1 shows a low viscosity morphology in submerged culture, enabling the use of complex growth and production media. C1 also does not "hyperglycosylate" heterologous proteins, as ''Aspergillus'' and ''Trichoderma'' tend to do.<ref name="Visser 214–223" />

===== ''Baculovirus''-infected cells =====
{{See also|BacMam}}
[[Baculoviridae|Baculovirus]]-infected insect cells<ref name="ReferenceA">{{cite journal | vauthors = Altmann F, Staudacher E, Wilson IB, März L | title = Insect cells as hosts for the expression of recombinant glycoproteins | journal = Glycoconjugate Journal | volume = 16 | issue = 2 | pages = 109–23 | date = February 1999 | pmid = 10612411 | doi = 10.1023/A:1026488408951 | s2cid = 34863069 }}</ref> ([[Sf9 (cells)|Sf9]], [[Sf21]], [[High Five cells|High Five]] strains) or mammalian cells<ref>{{cite journal | vauthors = Kost TA, Condreay JP | title = Recombinant baculoviruses as expression vectors for insect and mammalian cells | journal = Current Opinion in Biotechnology | volume = 10 | issue = 5 | pages = 428–33 | date = October 1999 | pmid = 10508635 | doi = 10.1016/S0958-1669(99)00005-1 }}</ref> ([[HeLa]], [[HEK 293 cells|HEK 293]]) allow production of glycosylated or membrane proteins that cannot be produced using fungal or bacterial systems.<ref name="ReferenceA"/><ref name="pmid15877075" /> 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.<ref>{{cite journal | vauthors = Yin J, Li G, Ren X, Herrler G | title = Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes | journal = Journal of Biotechnology | volume = 127 | issue = 3 | pages = 335–47 | date = January 2007 | pmid = 16959350 | doi = 10.1016/j.jbiotec.2006.07.012 }}</ref>

===== 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 [[Transfection|transfected]] into the chromosomal DNA of insect cells for subsequent gene expression.<ref name=Dyring>{{cite journal | last1 = Dyring | first1 = Charlotte | year = 2011 | title = Optimising the drosophila S2 expression system for production of therapeutic vaccines | journal = BioProcessing Journal | volume = 10 | issue = 2| pages = 28–35 | doi=10.12665/j102.dyring}}</ref><ref name=Olczak>{{cite journal | vauthors = Olczak M, Olczak T | title = Comparison of different signal peptides for protein secretion in nonlytic insect cell system | journal = Analytical Biochemistry | volume = 359 | issue = 1 | pages = 45–53 | date = December 2006 | pmid = 17046707 | doi = 10.1016/j.ab.2006.09.003 }}</ref> This is followed by selection and screening of recombinant clones.<ref name=McCarroll>{{cite journal | vauthors = McCarroll L, King LA | title = Stable insect cell cultures for recombinant protein production | journal = Current Opinion in Biotechnology | volume = 8 | issue = 5 | pages = 590–4 | date = October 1997 | pmid = 9353223 | doi = 10.1016/s0958-1669(97)80034-1 }}</ref> The non-lytic system has been used to give higher protein yield and quicker expression of recombinant genes compared to baculovirus-infected cell expression.<ref name=Olczak /> Cell lines used for this system include: [[Sf9 (cells)|Sf9]], [[Sf21]] from ''[[Spodoptera frugiperda]]'' cells, [[High Five cells|Hi-5]] from ''[[Cabbage looper|Trichoplusia ni]]'' cells, and [[Schneider 2 cells]] and Schneider 3 cells from ''[[Drosophila melanogaster]]'' cells.<ref name=Dyring /><ref name=McCarroll /> With this system, cells do not lyse and several cultivation modes can be used.<ref name=Dyring /> Additionally, protein production runs are reproducible.<ref name=Dyring /><ref name=Olczak /> This system gives a homogeneous product.<ref name=Olczak /> A drawback of this system is the requirement of an additional screening step for selecting viable [[Clone (cell biology)|clones]].<ref name=McCarroll />

===== ''[[Excavata]]'' =====
''[[Leishmania]] 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]] and disulfide bond formation.{{citation needed|date=August 2015}}

===== Mammalian systems =====
The most common mammalian expression systems are [[Chinese hamster|Chinese Hamster]] [[ovary]] (CHO) and Human embryonic kidney (HEK) cells.<ref name="Zhu 1158–1170">{{cite journal | vauthors = Zhu J | title = Mammalian cell protein expression for biopharmaceutical production | journal = Biotechnology Advances | volume = 30 | issue = 5 | pages = 1158–70 | date = 2012-09-01 | pmid = 21968146 | doi = 10.1016/j.biotechadv.2011.08.022 }}</ref><ref name=":0">{{cite journal | vauthors = Almo SC, Love JD | title = Better and faster: improvements and optimization for mammalian recombinant protein production | journal = Current Opinion in Structural Biology | volume = 26 | pages = 39–43 | date = June 2014 | pmid = 24721463 | pmc = 4766836 | doi = 10.1016/j.sbi.2014.03.006 | series = New constructs and expression of proteins / Sequences and topology }}</ref><ref>{{cite journal | vauthors = Hacker DL, Balasubramanian S | title = Recombinant protein production from stable mammalian cell lines and pools | journal = Current Opinion in Structural Biology | volume = 38 | pages = 129–36 | date = June 2016 | pmid = 27322762 | doi = 10.1016/j.sbi.2016.06.005 | series = New constructs and expression of proteins • Sequences and topology }}</ref>
* [[Chinese hamster ovary cell]]<ref name=":0" />
* [[Mouse]] myeloma [[Lymphoblast|lymphoblstoid]] (e.g. NS0 cell)<ref name="Zhu 1158–1170"/>
* Fully Human
** Human embryonic kidney cells ([[HEK 293 cells|HEK-293]])<ref name=":0" />
** Human embryonic retinal cells (Crucell's Per.C6)<ref name=":0" />
** Human [[amniocyte]] cells (Glycotope and CEVEC){{Cn|date=January 2024}}

===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.<ref>{{cite journal | vauthors = Rosenblum G, Cooperman BS | title = Engine out of the chassis: cell-free protein synthesis and its uses | journal = FEBS Letters | volume = 588 | issue = 2 | pages = 261–8 | date = January 2014 | pmid = 24161673 | pmc = 4133780 | doi = 10.1016/j.febslet.2013.10.016 | bibcode = 2014FEBSL.588..261R }}</ref>

== See also ==
* [[Cellosaurus]], a database of cell lines
* [[Gene expression]]
* [[Single-cell protein]]
* [[Protein purification]]
* [[Precision fermentation]]
* [[Host cell protein]]
* [[List of recombinant proteins]]

== References ==
{{Reflist|30em}}

== Further reading ==
* {{cite book|first1=Steve J. |last1=Higgins|first2=B. D. |last2=Hames | name-list-style = vanc |title=Protein Expression: A Practical Approach|url={{google books |plainurl=y |id=mQ7LOiVq4v0C}}|year=1999|publisher=Oxford University Press|isbn=978-0-19-963623-5}}
* {{cite book|first=François |last=Baneyx|title=Protein Expression Technologies: Current Status and Future Trends|url={{google books |plainurl=y |id=8YuHtwPFOXMC}}|year=2004|publisher=Garland Science|isbn=978-0-9545232-5-1}}

== External links ==
{{Library resources box}}

{{Microorganisms}}

[[Category:Gene expression]]
[[Category:Biotechnology]]