Fusion protein

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Chimeric antibodies
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This article is about chimeric fusion proteins. For proteins involved in membrane fusion, see membrane fusion protein.
A chimeric protein including two subunits and a linker protein synthesized via recombinant fusion technology

Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more

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
.

Functions

Some fusion proteins combine whole peptides and therefore contain all

functional 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 that alter when and where these genes act. For partial gene fusions, the shuffling of different active sites and binding domains have the potential to result in new proteins with novel functions.

Green fluorescent protein (GFP) inserted into the neurons of Caenorhabditis elegans worms to track neuronal development

Fluorescent protein tags

The fusion of

plasma membrane to be recycled, marked by a green fluorescent tag, or may be sent to a lysosome for degradation, marked by a red fluorescent tag.[1]

Chimeric protein drugs

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 is to impart properties from each of the "parent" proteins to the resulting chimeric protein. Several chimeric protein drugs are currently available for medical use.

Many chimeric protein drugs are

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.

tumor necrosis factor receptor (TNFR) with the immunoglobulin G1 Fc segment. TNFR provides specificity for the drug target and the antibody Fc segment is believed to add stability and deliverability of the drug.[2]
Additional chimeric proteins used for therapeutic applications include:

Recombinant technology

Fusion of two genes (BCR-ABL) to encode a recombinant oncogenic protein

A recombinant fusion protein is a

cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame through ligation or overlap extension PCR. That DNA sequence will then be expressed by a cell
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

antibodies attached to them in order to study disease development. Hydrogenase promoter, PSH, was studied constructing a PSH promoter-gfp fusion by using green fluorescent protein (gfp) reporter gene.[3]

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, and are known to increase the following properties:[4]

  • tertiary and quaternary structure of the target protein.[4]
  • 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.[4]
  • 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.[4]
  • Enzyme activity: Fusion that involves the introduction of hydrogen bonds may be used to expand overall enzyme activity.[4]
  • Expression levels: Addition of numerous fusion fragments, such as maltose binding protein (MBP) or small ubiquitin-like molecule (SUMO), serve to enhance enzyme expression and secretion of the target protein.[4]
  • Immobilization: PHA synthase, an enzyme that allows for the immobilization of proteins of interest, is an important fusion tag in industrial research.[4]
  • Crystal quality: Crystal quality can be improved by adding covalent links between proteins, aiding in structure determination techniques.[4]

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. 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.[5]

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. 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.[5]

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 site in the gene of interest.[5]

Post-translational conjugation

This technique fuses protein domains following ribosomal translation of the proteins of interest, in contrast to genetic fusion prior to translation used in other recombinant technologies.[5]

Protein linkers

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.[5][6]

  • Flexible linkers may consist of many small glycine residues, giving them the ability curl into a dynamic, adaptable shape.[6]
  • Rigid linkers may be formed of large, cyclic proline residues, which can be helpful when highly specific spacing between domains must be maintained.[6]
  • 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 gradient, or when coming in contact with another biomolecule in the cell.[6]

Natural occurrence

Naturally occurring fusion genes are most commonly created when a

spliced, and translated to produce a functional fusion protein. Many important cancer-promoting oncogenes
are fusion genes produced in this way.

Examples include:

Antibodies are fusion proteins produced by 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

Flavobacteria) and contain full-length cyclophilin and FKBP chaperone modules.[8][9]
The evolutionary origin of such chimera remains unclear.

See also

References

  1. PMID 24947379
    .
  2. ^
    S2CID 23852865
    .
  3. PMID 27547572
    .
  4. ^
    S2CID 14316893
    .
  5. ^
    PMID 25450191
    .
  6. ^
    PMID 23026637
    .
  7. PMID 9556615
    .
  8. PMID 15845546
    .
  9. PMID 28567569
    .

External links

Library resources about
Fusion protein
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