Fusion protein

Fusion protein

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Fusion proteins or chimeric proteins are proteins created through the joining of two or more gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

s which originally coded for separate proteins. Translation of this fusion gene
Fusion gene
A fusion gene is a hybrid gene formed from two previously separate genes. It can occur as the result of a translocation, interstitial deletion, or chromosomal inversion...

results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Chimeric mutant proteins occur naturally when a complex mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...

, such as a chromosomal translocation
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...

, 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 oncoproteins. The 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
Chronic myelogenous leukemia
Chronic myelogenous leukemia , also known as chronic granulocytic leukemia , is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood...

.

Functions


Some fusion proteins combine whole peptides and therefore contains all functional domains of the original proteins. However, other fusion proteins, especially those that are naturally occurring, 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
Regulation of gene expression
Gene modulation redirects here. For information on therapeutic regulation of gene expression, see therapeutic gene modulation.Regulation of gene expression includes the processes that cells and viruses use to regulate the way that the information in genes is turned into gene products...

 that alter when and where these genes act. For partial gene fusions
Chimeric gene
Chimeric genes form through the combination of portions of one or more coding sequences to produce new genes. These mutations are distinct from fusion genes which merge whole gene sequences into a single reading frame and often retain their original functions....

, the shuffling of different active sites and binding domains can potentially result in new proteins with novel functions.

Recombinant technology


A recombinant fusion protein is a protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 created through genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...

 of a fusion gene. This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame
Reading frame
In biology, a reading frame is a way of breaking a sequence of nucleotides in DNA or RNA into three letter codons which can be translated in amino acids. There are 3 possible reading frames in an mRNA strand: each reading frame corresponding to starting at a different alignment...

 through ligation
Ligase
In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...

 or overlap extension PCR. That DNA sequence will then be expressed
Protein expression
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...

 by a cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

 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
Protein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterization of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or...

, linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents which enable the liberation of the two separate proteins. This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide
FLAG-tag
FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism...

, or a hexa-his peptide (6xHis-tag) which can be isolated using affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...

 with nickel or cobalt resins. Fusion proteins can also be manufactured with toxin
Toxin
A toxin is a poisonous substance produced within living cells or organisms; man-made substances created by artificial processes are thus excluded...

s or antibodies attached to them in order to study disease development.

Chimeric protein drugs



The purpose of creating fusion proteins in drug development
Drug development
Drug development is a blanket term used to define the process of bringing a new drug to the market once a lead compound has been identified through the process of drug discovery...

 is to impart properties from each of the "parent" proteins to the resulting chimeric protein. Several chimeric protein drug
Drug
A drug, broadly speaking, is any substance that, when absorbed into the body of a living organism, alters normal bodily function. There is no single, precise definition, as there are different meanings in drug control law, government regulations, medicine, and colloquial usage.In pharmacology, a...

s are currently available for medical use.

Many chimeric protein drugs are monoclonal antibodies
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....

 whose specificity for a target molecule
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...

 was developed using mice and hence were initially "mouse" antibodies. As non-human proteins, mouse antibodies tend to evoke an immune reaction if administered to humans. The chimerization process involves engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...

 the replacement of segments of the antibody molecule that distinguish it from a human antibody. For example, human constant domains can be introduced, thereby eliminating most of the potentially immunogenic
Immunogenicity
Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune response in the body of a human or animal.- Immunogenicity :The ability to induce humoral and/or cell-mediated immune responses....

 portions of the drug without altering its specificity for the intended therapeutic target. Antibody nomenclature indicates this type of modification by inserting -xi- into the non-proprietary name
International Nonproprietary Name
An International Nonproprietary Name is the official nonproprietary or generic name given to a pharmaceutical substance, as designated by the World Health Organization...

 (e.g. abci-xi-mab). If parts of the variable domains are also replaced by human portions, humanized antibodies are obtained. Although not conceptually distinct from chimeras, this type is indicated using -zu- such as in dacli-zu-mab
Daclizumab
Daclizumab is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants....

. See the 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
Etanercept
Etanercept is a drug that treats autoimmune diseases by interfering with the tumor necrosis factor by acting as a TNF inhibitor. Pfizer describes in a SEC filing that the drug is used to treat rheumatoid, juvenile rheumatoid and psoriatic arthritis, plaque psoriasis and ankylosing spondylitis...

, for example, is a TNFα blocker created through the combination of a tumor necrosis factor receptor
Tumor necrosis factor receptor
A tumor necrosis factor receptor , or death receptor, is a trimeric cytokine receptor that binds tumor necrosis factors . The receptor cooperates with an adaptor protein , which is important in determining the outcome of the response A tumor necrosis factor receptor (TNFR), or death receptor, is a...

 (TNFR) with the immunoglobulin G
Immunoglobulin G
Immunoglobulin G are antibody molecules. Each IgG is composed of four peptide chains — two heavy chains γ and two light chains. Each IgG has two antigen binding sites. Other immunoglobulins may be described in terms of polymers with the IgG structure considered the monomer.IgG constitutes 75%...

1 Fc segment
Fragment crystallizable region
The fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system...

. TNFR provides specificity for the drug target and the antibody Fc segment is believed to add stability and deliverability of the drug.

Natural occurrence


Naturally occurring fusion genes are most commonly created when a chromosomal
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...

 translocation
Translocation
Translocation may refer to:* Chromosomal translocation, in genetics* Translocation in plants, transport of food or pesticides through phloem or xylem* Protein translocation or protein targeting, a process in protein biosynthesis...

 replaces the terminal exon
Exon
An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule either after portions of a precursor RNA have been removed by cis-splicing or when two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA...

s of one gene with intact exons from a second gene. This creates a single gene which can be transcribed
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...

, spliced
Splicing (genetics)
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...

, and translated to produce a functional fusion protein. Many important cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

-promoting oncogene
Oncogene
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.An oncogene is a gene found in the chromosomes of tumor cells whose activation is associated with the initial and continuing conversion of normal cells into cancer...

s are fusion genes produced in this way.

Examples include:
  • Gag-onc fusion protein
    Gag-onc fusion protein
    The gag-onc fusion protein is a protein formed from a group-specific antigen and that of an oncovirus , such as C-jun....

  • Bcr-abl fusion protein
  • Tpr-met fusion protein
    Tpr-met fusion protein
    Tpr-met fusion protein is an oncogene fusion protein consisting of TPR and MET....



Antibodies are fusion proteins produced by VDJ recombination.