# Fusion protein

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{{Short description|Protein created by joining other proteins into a single polypeptide}}
{{About|chimeric fusion proteins|proteins involved in membrane fusion|membrane fusion protein}}
thumb|331x331px|A chimeric protein including two subunits and a linker protein synthesized via recombinant fusion technology
'''Fusion proteins''' or '''chimeric proteins''' (literally, made of parts from different sources) are proteins created through the joining of two or more [gene](/source/gene)s that originally coded for separate proteins. Translation of this ''[fusion gene](/source/fusion_gene)'' results in a single or multiple [polypeptides](/source/polypeptides) with functional properties derived from each of the original proteins. ''Recombinant fusion proteins'' are created artificially by [recombinant DNA technology](/source/recombinant_DNA_technology) for use in biological research or [therapeutics](/source/therapeutics). ''[Chimeric](/source/Chimera_(genetics))'' or ''chimera'' usually designate hybrid proteins made of [polypeptides](/source/polypeptides) having different functions or physico-chemical patterns. ''Chimeric mutant proteins'' occur naturally when a complex [mutation](/source/mutation), such as a [chromosomal translocation](/source/chromosomal_translocation), tandem duplication, or retrotransposition creates a novel coding sequence containing parts of the coding sequences from two different genes. Naturally occurring fusion proteins are commonly found in cancer cells, where they may function as [oncoprotein](/source/oncoprotein)s. The [bcr-abl fusion protein](/source/bcr-abl_fusion_protein) is a well-known example of an oncogenic fusion protein, and is considered to be the primary oncogenic driver of [chronic myelogenous leukemia](/source/chronic_myelogenous_leukemia).

In the [International nonproprietary name](/source/International_nonproprietary_name) scheme, drugs based on fusion proteins are given the ''-fusp'' suffix.<ref>{{cite web |title=INN for fusion protein |url=https://www.who.int/publications/i/item/inn-17-414 |website=www.who.int |language=en}}</ref>

==Functions==
Some fusion proteins combine whole peptides and therefore contain all [functional domains](/source/Protein_domains) of the original proteins.  However, other fusion proteins, especially those that occur naturally, combine only portions of coding sequences and therefore do not maintain the original functions of the parental genes that formed them.

Many whole gene fusions are fully functional and can still act to replace the original peptides. Some, however, experience interactions between the two proteins that can modify their functions.  Beyond these effects, some gene fusions may cause [regulatory changes](/source/Regulation_of_gene_expression) that alter when and where these genes act. For [partial gene fusions](/source/Chimeric_gene), the shuffling of different active sites and binding domains have the potential to result in new proteins with novel functions.
[[File:Varbuss.jpg|thumb|263x263px|Green fluorescent protein (GFP) inserted into the neurons of ''[Caenorhabditis elegans](/source/Caenorhabditis_elegans)'' worms to track neuronal development]]

=== Fluorescent protein tags ===
The fusion of [fluorescent tag](/source/fluorescent_tag)s to proteins in a host cell is a widely popular technique used in experimental cell and biology research in order to track protein interactions in real time. The first fluorescent tag, [green fluorescent protein](/source/green_fluorescent_protein) (GFP), was isolated from ''[Aequorea victoria](/source/Aequorea_victoria)'' and is still used frequently in modern research. More recent derivations include photoconvertible fluorescent proteins (PCFPs), which were first isolated from ''[Anthozoa](/source/Anthozoa)''. The most commonly used PCFP is the [Kaede](/source/Kaede_(protein)) fluorescent tag, but the development of Kikume green-red (KikGR) in 2005 offers a brighter signal and more efficient photoconversion. The advantage of using PCFP fluorescent tags is the ability to track the interaction of overlapping biochemical pathways in real time. The tag will change color from green to red once the protein reaches a point of interest in the pathway, and the alternate colored protein can be monitored through the duration of pathway. This technique is especially useful when studying [G-protein coupled receptor](/source/G_protein%E2%80%93coupled_receptor) (GPCR) recycling pathways. The fates of recycled G-protein receptors may either be sent to the [plasma membrane](/source/plasma_membrane) to be recycled, marked by a green fluorescent tag, or may be sent to a [lysosome](/source/lysosome) for degradation, marked by a red fluorescent tag.<ref>{{Cite book|journal=<!--Bypass Citation bot -->|volume=1174|last1=Schmidt|first1=Antje|last2=Wiesner|first2=Burkhard|last3=Schülein|first3=Ralf|last4=Teichmann|first4=Anke |title=Exocytosis and Endocytosis |chapter=Use of Kaede and Kikume Green–Red Fusions for Live Cell Imaging of G Protein-Coupled Receptors | name-list-style = vanc |publisher=Humana Press|year=2014|isbn=978-1-4939-0943-8|series=Methods in Molecular Biology|location=New York, NY|pages=139–156|language=en|doi=10.1007/978-1-4939-0944-5_9|pmid = 24947379}}</ref>

===Chimeric protein drugs===
[[File:Chimeric and humanized antibodies.svg|thumb|259x259px|Sketches of mouse (top-left), chimeric (top-right) and [humanized](/source/humanized) (bottom-left) monoclonal antibodies. Human parts are shown in brown, and non-human parts in blue.]]
The purpose of creating fusion proteins in [drug development](/source/drug_development) is to impart properties from each of the "parent" proteins to the resulting chimeric protein. Several chimeric protein [drug](/source/drug)s are currently available for medical use.

Many chimeric protein drugs are [monoclonal antibodies](/source/monoclonal_antibodies) whose specificity for a [target molecule](/source/antigen) was developed using mice and hence were initially "mouse" antibodies. As non-human proteins, mouse antibodies tend to evoke an [immune reaction](/source/immune_reaction) if administered to humans. The chimerization process involves [engineering](/source/genetic_engineering) the replacement of segments of the antibody molecule that distinguish it from a human antibody. For example, human [constant domain](/source/constant_domain)s can be introduced, thereby eliminating most of the potentially [immunogenic](/source/immunogenicity) portions of the drug without altering its specificity for the intended therapeutic target. [Antibody nomenclature](/source/Antibody_nomenclature) indicates this type of modification by inserting ''-xi-'' into the [non-proprietary name](/source/International_Nonproprietary_Name) (e.g., [abci-''xi''-mab](/source/Abciximab)). If parts of the variable domains are also replaced by human portions, ''[humanized](/source/humanized)'' antibodies are obtained. Although not conceptually distinct from chimeras, this type is indicated using ''-zu-'' such as in [dacli-''zu''-mab](/source/Daclizumab). See the [list of monoclonal antibodies](/source/list_of_monoclonal_antibodies) for more examples.

In addition to chimeric and humanized antibodies, there are other pharmaceutical purposes for the creation of chimeric constructs. [Etanercept](/source/Etanercept), for example, is a [TNFα](/source/Tumor_necrosis_factor-alpha) blocker created through the combination of a [tumor necrosis factor receptor](/source/tumor_necrosis_factor_receptor) (TNFR) with the [immunoglobulin G](/source/immunoglobulin_G)1 [Fc segment](/source/Fragment_crystallizable_region). TNFR provides specificity for the drug target and the antibody Fc segment is believed to add stability and deliverability of the drug.<ref name=":0" /> Additional chimeric proteins used for therapeutic applications include:
* [Aflibercept](/source/Aflibercept): A human recombinant protein that aids in the treatment of oxaliplatin-resistant [metastatic colorectal cancer](/source/metastatic_colorectal_cancer), neo-vascular [macular degeneration](/source/macular_degeneration), and [macular edema](/source/macular_edema).<ref name=":0" />
* [Rilonacept](/source/Rilonacept): Reduces inflammation by preventing activation of [IL-1 receptors](/source/IL1_receptor_family) to treat [cryopyrin-associated periodic syndrome](/source/cryopyrin-associated_periodic_syndrome)s (CAPS).<ref name=":0" />
* [Alefacept](/source/Alefacept): Regulated [T-cell](/source/T_cell) responses by selectively targeting effector memory T-cells to treat [psoriasis vulgaris](/source/psoriasis_vulgaris).<ref name=":0" />
* [Romiplostim](/source/Romiplostim): A peptibody that treats [immune thrombocytopenia](/source/immune_thrombocytopenia).<ref name=":0" />
* [Abatacept](/source/Abatacept)/[Belatacept](/source/Belatacept): Interferes with T-cell co-stimulation to treat autoimmune disorders like [rheumatoid arthritis](/source/rheumatoid_arthritis), [psoriatic arthritis](/source/psoriatic_arthritis), and [psoriasis](/source/psoriasis).<ref name=":0" />
* [Denileukin-diftitox](/source/Denileukin_diftitox): Treats [cutaneous lymphoma](/source/cutaneous_lymphoma).<ref name=":0">{{cite journal | vauthors = Baldo BA | title = Chimeric fusion proteins used for therapy: indications, mechanisms, and safety | journal = Drug Safety | volume = 38 | issue = 5 | pages = 455–79 | date = May 2015 | pmid = 25832756 | doi = 10.1007/s40264-015-0285-9 | s2cid = 23852865 }}</ref>

==Recombinant technology==
thumb|399x399px|Fusion of two genes (BCR-ABL) to encode a recombinant oncogenic protein
A '''recombinant fusion protein''' is a [protein](/source/protein) created through [genetic engineering](/source/genetic_engineering) of a fusion gene. This typically involves removing the stop [codon](/source/codon) from a [cDNA](/source/cDNA) sequence coding for the first protein, then appending the cDNA sequence of the second protein [in frame](/source/reading_frame) through [ligation](/source/ligase) or [overlap extension PCR](/source/Overlap_extension_polymerase_chain_reaction).  That DNA sequence will then be [expressed](/source/gene_expression) by a [cell](/source/cell_(biology)) as a single protein. The protein can be engineered to include the full sequence of both original proteins, or only a portion of either.

If the two entities are proteins, often linker (or "spacer") peptides are also added, which make it more likely that the proteins fold independently and behave as expected.  Especially in the case where the linkers enable [protein purification](/source/protein_purification), linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents that enable the liberation of the two separate proteins. This technique is often used for identification and purification of proteins, by fusing a [GST protein](/source/GST-tag), [FLAG peptide](/source/FLAG-tag), or a [hexa-his peptide](/source/his-tag) (6xHis-tag), which can be isolated using [affinity chromatography](/source/affinity_chromatography) with nickel or cobalt resins. Di- or multimeric chimeric proteins can be manufactured through [genetic engineering](/source/genetic_engineering) by fusion to the original proteins of peptide domains that induce artificial protein di- or multimerization (e.g., [streptavidin](/source/streptavidin) or [leucine zippers](/source/leucine_zippers)). Fusion proteins can also be manufactured with [toxin](/source/toxin)s or [antibodies](/source/antibodies) attached to them in order to study disease development. Hydrogenase promoter, P<sub>SH</sub>, was studied constructing a P<sub>SH</sub> promoter-''gfp'' fusion by using [green fluorescent protein (''gfp)''](/source/Green_fluorescent_protein) [reporter gene](/source/reporter_gene).<ref>{{cite journal | vauthors = Jugder BE, Welch J, Braidy N, Marquis CP | title = Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein) | journal = PeerJ | volume = 4 | article-number = e2269 | date = 2016-07-26 | pmid = 27547572 | pmc = 4974937 | doi = 10.7717/peerj.2269 | doi-access = free }}</ref>

=== Recombinant functionality ===
Novel recombinant technologies have made it possible to improve fusion protein design for use in fields as diverse as biodetection, paper and food industries, and biopharmaceuticals. Recent improvements have involved the fusion of single peptides or protein fragments to regions of existing proteins, such as [N and C termini](/source/N-terminus), and are known to increase the following properties:<ref name=":1" /> 
* [Catalytic efficiency](/source/Catalytic_efficiency): Fusion of certain peptides allow for greater catalytic efficiency by altering the [tertiary](/source/Tertiary_structure) and [quaternary structure](/source/Biomolecular_structure) of the target protein.<ref name=":1" />
* [Solubility](/source/Solubility): A common challenge in fusion protein design is the issue of insolubility of newly synthesized fusion proteins in the recombinant host, leading to an over-aggregation of the target protein in the cell. [Molecular chaperones](/source/Molecular_chaperones) that are able to aid in protein folding may be added, thereby better segregating hydrophobic and hydrophilic interactions in the solute to increase protein solubility.<ref name=":1" />
* [Thermostability](/source/Thermostability): Singular peptides or protein fragments are typically added to reduce flexibility of either the N or C terminus of the target protein, which reinforces thermostability and stabilizes [pH range](/source/PH).<ref name=":1" />
* Enzyme activity: Fusion that involves the introduction of [hydrogen bond](/source/hydrogen_bond)s may be used to expand overall enzyme activity.<ref name=":1" /> 
* Expression levels: Addition of numerous fusion fragments, such as [maltose binding protein](/source/Maltose-binding_protein) (MBP) or small ubiquitin-like molecule ([SUMO](/source/SUMO_protein)), serve to enhance enzyme expression and secretion of the target protein.<ref name=":1" /> 
* Immobilization: PHA synthase, an enzyme that allows for the immobilization of proteins of interest, is an important fusion tag in industrial research.<ref name=":1" /> 
* Crystal quality: Crystal quality can be improved by adding covalent links between proteins, aiding in structure determination techniques.<ref name=":1">{{cite journal | vauthors = Yang H, Liu L, Xu F | title = The promises and challenges of fusion constructs in protein biochemistry and enzymology | journal = Applied Microbiology and Biotechnology | volume = 100 | issue = 19 | pages = 8273–81 | date = October 2016 | pmid = 27541749 | doi = 10.1007/s00253-016-7795-y | s2cid = 14316893 }}</ref>

== Recombinant protein design ==
The earliest applications of recombinant protein design can be documented in the use of single peptide tags for purification of proteins in [affinity chromatography](/source/affinity_chromatography). Since then, a variety of fusion protein design techniques have been developed for applications as diverse as fluorescent protein tags to recombinant fusion protein drugs. Three commonly used design techniques include tandem fusion, domain insertion, and post-translational conjugation.<ref name=":2">{{cite journal | vauthors = Yu K, Liu C, Kim BG, Lee DY | title = Synthetic fusion protein design and applications | journal = Biotechnology Advances | volume = 33 | issue = 1 | pages = 155–164 | date = 2015-01-01 | pmid = 25450191 | doi = 10.1016/j.biotechadv.2014.11.005 | bibcode = 2015BiotA..33..155Y }}</ref>

=== Tandem fusion ===

The proteins of interest are simply connected end-to-end via fusion of N or C termini between the proteins. This provides a flexible bridge structure allowing enough space between fusion partners to ensure proper [folding](/source/Protein_folding). However, the N or C termini of the peptide are often crucial components in obtaining the desired folding pattern for the recombinant protein, making simple end-to-end conjoining of domains ineffective in this case. For this reason, a protein linker is often needed to maintain the functionality of the protein domains of interest.<ref name=":2" />

=== Domain insertion ===

This technique involves the fusion of consecutive protein domains by encoding desired structures into a single polypeptide chain, but sometimes may require insertion of a domain within another domain. This technique is typically regarding as more difficult to carry out than tandem fusion, due to difficulty finding an appropriate [ligation](/source/Ligation_(molecular_biology)) site in the gene of interest.<ref name=":2" />

=== Post-translational conjugation ===

This technique fuses protein domains following ribosomal [translation](/source/Translation_(biology)) of the proteins of interest, in contrast to genetic fusion prior to translation used in other recombinant technologies.<ref name=":2" />

=== Protein linkers ===
thumb|A protein used as a linker in fusion protein design
Protein linkers aid fusion protein design by providing appropriate spacing between domains, supporting correct protein folding in the case that N or C termini interactions are crucial to folding. Commonly, protein linkers permit important domain interactions, reinforce stability, and reduce steric hindrance, making them preferred for use in fusion protein design even when N and C termini can be fused. Three major types of linkers are flexible, rigid, and in vivo cleavable.<ref name=":2" /><ref name=":3">{{cite journal | vauthors = Chen X, Zaro JL, Shen WC | title = Fusion protein linkers: property, design and functionality | journal = Advanced Drug Delivery Reviews | volume = 65 | issue = 10 | pages = 1357–69 | date = October 2013 | pmid = 23026637 | pmc = 3726540 | doi = 10.1016/j.addr.2012.09.039 }}</ref> 
* '''Flexible linkers''' may consist of many small [glycine](/source/glycine) residues, giving them the ability curl into a dynamic, adaptable shape.<ref name=":3" /> 
* '''Rigid linkers''' may be formed of large, cyclic [proline](/source/proline) residues, which can be helpful when highly specific spacing between domains must be maintained.<ref name=":3" /> 
* '''''In vivo'' cleavable linkers''' are unique in that they are designed to allow the release of one or more fused domains under certain reaction conditions, such as a specific [pH](/source/pH) gradient, or when coming in contact with another [biomolecule](/source/biomolecule) in the cell.<ref name=":3" />

==Natural occurrence==
Naturally occurring fusion genes are most commonly created when a [chromosomal translocation](/source/chromosomal_translocation) replaces the terminal [exon](/source/exon)s of one gene with intact exons from a second gene. This creates a single gene that can be [transcribed](/source/transcription_(genetics)), [spliced](/source/splicing_(genetics)), and [translated](/source/translation_(biology)) to produce a functional fusion protein. Many important [cancer](/source/cancer)-promoting [oncogene](/source/oncogene)s are fusion genes produced in this way.

Examples include:
* [Gag-onc fusion protein](/source/Gag-onc_fusion_protein)
* [Bcr-abl fusion protein](/source/Bcr-abl_fusion_protein)
* [Tpr-met fusion protein](/source/Tpr-met_fusion_protein)

Antibodies are fusion proteins produced by [V(D)J recombination](/source/V(D)J_recombination).

There are also rare examples of naturally occurring polypeptides that appear to be a fusion of two clearly defined modules, in which each module displays its characteristic activity or function, independent of the other. Two major examples are: double PP2C chimera in ''[Plasmodium falciparum](/source/Plasmodium_falciparum)'' (the malaria parasite), in which each PP2C module exhibits protein phosphatase 2C enzymatic activity,<ref>{{cite journal | vauthors = Mamoun CB, Sullivan DJ, Banerjee R, Goldberg DE | title = Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum | journal = The Journal of Biological Chemistry | volume = 273 | issue = 18 | pages = 11241–7 | date = May 1998 | pmid = 9556615 | doi = 10.1074/jbc.273.18.11241 | doi-access = free }}</ref> and the [dual-family immunophilin](/source/dual-family_immunophilin)s that occur in a number of unicellular organisms (such as protozoan parasites and [Flavobacteria](/source/Flavobacteria)) and contain full-length [cyclophilin](/source/cyclophilin) and [FKBP](/source/FKBP) chaperone modules.<ref>{{cite journal | vauthors = Adams B, Musiyenko A, Kumar R, Barik S | title = A novel class of dual-family immunophilins | journal = The Journal of Biological Chemistry | volume = 280 | issue = 26 | pages = 24308–14 | date = July 2005 | pmid = 15845546 | pmc = 2270415 | doi = 10.1074/jbc.M500990200 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Barik S | title = On the role, ecology, phylogeny, and structure of dual-family immunophilins | journal = Cell Stress & Chaperones | volume = 22 | issue = 6 | pages = 833–845 | date = November 2017 | pmid = 28567569 | pmc = 5655371 | doi = 10.1007/s12192-017-0813-x }}</ref> The evolutionary origin of such chimera remains unclear.

== See also ==
*[Genetic engineering](/source/Genetic_engineering)
*[Protein engineering](/source/Protein_engineering)
*[Cell–cell fusogens](/source/Cell%E2%80%93cell_fusogens)

== References ==
{{Reflist}}

== External links ==
{{Library resources box
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* {{MeshName|Mutant+Chimeric+Proteins}}
* [http://chippi.md.biu.ac.il/ ChiPPI] {{Webarchive|url=https://web.archive.org/web/20211110060245/http://chippi.md.biu.ac.il/ |date=2021-11-10 }}: The Server Protein–Protein Interaction of Chimeric Proteins.

Category:Engineered proteins

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Adapted from the Wikipedia article [Fusion protein](https://en.wikipedia.org/wiki/Fusion_protein) by Wikipedia contributors ([contributor history](https://en.wikipedia.org/wiki/Fusion_protein?action=history)). Available under [Creative Commons Attribution-ShareAlike 4.0 International](https://creativecommons.org/licenses/by-sa/4.0/). Changes may have been made.