Elastin-like polypeptides

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Depicted above is an ELP monomeric unit, in which the X residue here is a threonine. From this monomeric unit, the ELP polymer would be created. General Structure ELP.png
Depicted above is an ELP monomeric unit, in which the X residue here is a threonine. From this monomeric unit, the ELP polymer would be created.

Elastin-like polypeptides (ELPs) are synthetic biopolymers with potential applications in the fields of cancer therapy, tissue scaffolding, metal recovery, and protein purification. For cancer therapy, the addition of functional groups to ELPs can enable them to conjugate with cytotoxic drugs. [1] Also, ELPs may be able to function as polymeric scaffolds, which promote tissue regeneration. This capacity of ELPs has been studied particularly in the context of bone growth. [2] ELPs can also be engineered to recognize specific proteins in solution. The ability of ELPs to undergo morphological changes at certain temperatures enables specific proteins that are bound to the ELPs to be separated out from the rest of the solution via experimental techniques such as centrifugation. [3]

Contents

The general structure of polymeric ELPs is (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (Tt). Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue play a great role in determining the Tt. Also, the solubilization of the guest residue can effect the Tt. The "n" denotes the number of monomeric units that comprise the polymer. [4] [5] [6] [7] In general, these polymers are linear below the Tt, but aggregate into spherical clumps above the Tt.. [3]

Structure

Although engineered and modified in a laboratory setting, ELPs share structural characteristics with intrinsically disordered proteins (IDPs) naturally found in the body, such as tropoelastin, from which ELPs were given their name. The repeat sequences found in the biopolymer give each ELP a distinct structure, as well as influence the lower critical solution temperature (LCST), also referred to commonly as the Tt. It is at this temperature that the ELPs move from a linear, relatively disordered state to a more densely aggregated, partially ordered state [7] Although given as a single temperature, Tt, the ELP phase change process generally begins and ends within a temperature range of approximately 2 °C. Also, Tt is altered by the addition of unique proteins to the free ELPs. [5]

Tropoelastin

This image displays the mechanism by which lysine residues on tropoelastin are cross-linked together. Conversion of some lysine residues to allysine occurs first, followed by bonding between lysine and allysine. This enables the elastin to form in the extracellular matrix. Lox mechanism.jpg
This image displays the mechanism by which lysine residues on tropoelastin are cross-linked together. Conversion of some lysine residues to allysine occurs first, followed by bonding between lysine and allysine. This enables the elastin to form in the extracellular matrix.

Tropoelastin is a protein, of size 72kDa, that comes together via cross-links to form elastin in the extracellular matrix of the cell. The cross-link formation process is mediated by lysyl oxidase. [8] One of the major reasons that elastin can withstand high levels of stress in the body without experiencing any physical deformation is that the underlying tropoelastin contains domains that are highly hydrophobic. These hydrophobic domains, consisting overwhelmingly of alanine, proline, glycine, and valine, tend towards instability and disorderliness, ensuring that the elastin does not lock into any specific confirmation. Thus, ELPs consisting of the Val-Pro-Gly-X-Gly monomeric units, which bear resemblance to the repetitive tropoelastin hydrophobic domains, are highly disordered below their Tt. Even above their Tt in their aggregated state, ELPs are only partially ordered. This is due to the fact that the proline and glycine amino acids are present in high amounts in the ELP. Glycine, due to the lack of a bulky side chain, enables the biopolymer to be flexible and proline prevents the formation of stable hydrogen bonds in the ELP backbone. It is important to note, however, that certain segments of the ELP may be able to form instantaneous type II β turns, but these turns are not long-lasting and do not resemble true β sheets, when the NMR chemical shifts are compared. [7]

This video depicts the assembly of tropoelastin units to form elastin.

Amyloid formation

Although ELPs generally form reversible spherical aggregates due to their proline and glycine content, there is a possibility that, under certain conditions such as exceedingly high temperatures, ELPs will form amyloids, or irreversible aggregates of insoluble protein. It is also believed that changes in the ELP backbone leading to a reduction in the proline and glycine content may lead to ELPs with a greater propensity for the amyloid state. As amyloids are implicated in the progression of Alzheimer's disease as well as in prion-based diseases, such as Creutzfeldt-Jakob disease (CJD), modeling of ELP amyloid formation may be useful from a biomedical standpoint. [7]

Tt dependence on ELP structure

The transition temperature of an ELP depends to a certain extent on the identity of the "X" residue found at the fourth position of the pentapeptide monomeric unit. Residues that are highly hydrophobic, such as leucine and phenylalanine, tend to decrease the transition temperature. On the other hand, residues that are highly hydrophilic, such as serine and glutamine, tend to increase the transition temperature. The presence of a potentially charged residue at the "X" position will determine how the ELP responds to varying pHs, with glutamic acid and aspartic acid raising the Tt at pH values in which the residues are deprotonated and lysine and arginine raising the Tt at pH values in which the residues are protonated. The pH needs to be compatible with the charged states of these amino acids in order to raise the Tt. Also higher molecular mass ELPs and higher concentrations of ELPs in solution make it much easier for the polymer to form aggregates, in effect lowering the experimental Tt. [9]

Tt theoretical model

Oftentimes, ELPs are not used in isolation, but are rather fused with other proteins to become functionally active. The structure of these other proteins will have a certain effect on transition temperature. It is important to be able to predict the transition temperature that these fusion proteins will have relative to the free ELPs, as this temperature will determine the fused protein's applicability and phase transition. A theoretical model is available that relates the change in Tt of the fused protein to the varying ratios of each individual amino acid found in the fused protein. The model involves calculating a surface index (SI) associated with each amino acid and then extrapolating, based on the ratio of each amino acid present in the fused protein, the total change in the Tt associated with the fusion protein, ΔTt,fusion: [10]

SI=(ASAXAA/ ASAp)(Ttc) [10]

where ASAp refers to the area of the entire fused protein that is available to the solvent that is being used, ASAXAA refers to the area of the guest residue on the ELP that is available to the solvent, and Ttc is the transition temperature that is unique to the amino acid. Summing up the contribution of each potential guest residue (XAA) will yield an SI index that is directly proportional to ΔTt,fusion. It was found that the amino acids that are charged under a physiological pH of 7.4 have the greatest impact on the overall SI of a fused protein. This is due to the fact that they are more accessible to water-containing solvents, thereby increasing the ASAXAA and also have high Ttc values. Hence, knowledge of the transition temperature of a fused protein is highly dependent on the presence of these charged residues. [10]

Synthesis

Because ELPs are protein-based biopolymers, synthesis involves manipulation of genes to continually express the monomeric repeat unit. Various techniques have been employed in the production of ELPs of various sizes, including unidirectional ligation or concatemerization, overlap extension polymerase chain reaction (OEPCR), and recursive directional ligation (RDL). [5] [9] Also, ELPs can be experimentally modified through conjugation with other polymers or through SpyTag/SpyCatcher reaction, [11] allowing for the synthesis of copolymers with unique morphology. [12]

Concatemerization

The concatemerization process generates libraries of concatamers for the ELPs. Concatamers are oligomeric products of ligating a single gene with itself. This will result in repeat segments of a gene, all of which can be transcribed and translated immediately to produce the ELP of interest. A major problem with this synthetic route is that the number of gene repeat segments ligated together to form the concatamer cannot be controlled, leading to ELPs of different sizes, from which the ELP of a desired size must be isolated. [9]

Overlap extension polymerase chain reaction (OEPCR)

The OEPCR method uses a small amount of the gene encoding the monomeric ELP unit and leads to the amplification of this segment to a great extent. This amplification is due to the fact that the initial segment added to the reaction functions as a template, from which identical gene segments can be synthesized. The process will result in the production of double-stranded DNA encoding the ELP of interest. One major bottleneck associated with this method is the potentially low fidelity associated with the Taq polymerase used. This might lead to replication from the template in which the wrong nucleotides are incorporated into the growing DNA strand. [9]

Recursive directional ligation (RDL)

In recursive directional ligation, the gene encoding the monomer is inserted into a plasmid with restriction sites that are recognized by at least two endonucleases. The endonucleases will cut the plasmid, releasing the gene of interest. Then, this single gene is inserted into a recipient plasmid vector already containing one copy of the ELP monomer gene via digestion of the recipient plasmid with the same restriction endonucleases used on the donor plasmid and a subsequent ligation step. From this process, a sequence of two ELP monomer genes is retrieved. RDL allows for the controlled synthesis of ELP gene oligomers, in which single gene segments are sequentially added. However, the restriction endonucleases used are limited to those that do not cut within the ELP monomer gene itself, as this would lead to loss of crucial nucleotides and a potential frameshift mutation in the protein. [5]

Synthetic conjugation

ELPs can be synthetically conjugated to poly (ethylene glycol) by adding a cyclooctyne functional motif to the poly (ethylene glycol) and an azide group to the ELP. Through a cycloaddition reaction involving both of the functional groups and manipulation of the solvent pH, diblock and star polymers can be formed. Rather than forming the canonical spherical clumps above the transition temperature, this specific conjugated ELP forms a micelle with amphiphillic properties, in which the polar head groups face outward and the hydrophobic domains face inward. Such micelles may be helpful in delivering nonpolar drugs to the body. [12]

Applications

Due to the unique temperature-dependent phase transition experienced by ELPs, in which they move from a linear state to a spherical aggregate state above their Tt, as well as the ability of ELPs to be easily conjugated with other compounds, these biopolymers hold numerous applications. Some of these applications involve ELP use in protein purification, cancer therapy, and tissue scaffolding. [1] [2] [3]

Protein purification

This diagram shows how proteins can isolated using ELP technology. At temperatures below the transition temperature, the ELP remains in its linear state but binds to the protein of interest via a functional group. As the solution is heated above the transition temperature, the ELP will start to form spherical clumps that will aggregate at the bottom of the tube following centrifugation. The ELP will contain the protein of interest (blue) and separate it from extraneous proteins (purple). Revised Protein Purification.png
This diagram shows how proteins can isolated using ELP technology. At temperatures below the transition temperature, the ELP remains in its linear state but binds to the protein of interest via a functional group. As the solution is heated above the transition temperature, the ELP will start to form spherical clumps that will aggregate at the bottom of the tube following centrifugation. The ELP will contain the protein of interest (blue) and separate it from extraneous proteins (purple).

The ELP can be conjugated to a functional group that can bind to a protein of interest. At temperatures below the Tt, the ELP will bind to the ligand in its linear form. In this linear state, the ELP-protein complex cannot easily be distinguished from the extraneous proteins in the solution. However, once the solution is heated to a temperature exceeding the Tt, the ELP will form spherical clumps. These clumps will then settle to the bottom of the solution tube following centrifugation, carrying the protein of interest. The proteins that are not needed will be found in the supernatant, which can be physically separated from the spherical aggregates. To ensure that there are few impurities in the ELP-protein complex isolated, the solution can be cooled below the Tt, enabling the ELPs to once again assume their linear structure. From this point, hot and cold centrifugation cycles can be repeated, and then the protein of interest can be eluted from the ELPs via the addition of a salt. [3]

Tissue scaffolding

The temperature-based phase behavior of ELPs can be utilized to produce stiff networks that may be compatible with cellular regeneration applications. At high concentrations (weight percent exceeding 15%), the ELP transition from a linear state to a spherical aggregate state above the transition temperature is arrested, leading to the formation of brittle gels. These otherwise brittle networks can then be modified chemically, via oxidative coupling, to yield hydrogels which can sustain high levels of mechanical stress and strain. Also, the modified gel networks contain pores, through which important cell-sustaining compounds can easily be delivered. Such strong hydrogels, when bathed in minimal cell media, have been found to promote the growth of human mesencyhmal stem cell populations. The ability of these arrested ELP networks to promote cell growth may prove indispensable in the production of tissue scaffolds that promote cartilage production, for example. Such an intervention may prove useful in the treatment of bone disease and rheumatoid arthritis. [2]

Drug delivery

Depicted above is the chemotherapeutic agent doxorubicin conjugated with ELPs. Chemotherapeutic Agent.png
Depicted above is the chemotherapeutic agent doxorubicin conjugated with ELPs.

ELPs modified with certain functional groups have the capacity to be conjugated with drugs, including chemotherapeutic agents. [13] Together, the ELP-drug complex can be taken up by tumor cells to a greater extent, promoting the cytotoxic activity of the drug. The reason that the complexes preferentially target the tumor cells is that these cells tend to be associated with more permeable blood vessels and also possess a weaker lymphatic presence. This essentially means that the drugs can cross over from the vessels to the tumor cells more frequently and can remain in the vessels for a longer period of time, without being filtered out. The phase transition associated with ELPs can also be used to promote tumor cell uptake of the drug. By locally heating tumor cell regions, the ELP-drug complex will aggregate into spherical clumps. If this ELP-drug complex is engineered to expose functional domains in the spherical clump shape that are recognized by tumor cell surfaces, then this cell surface interaction would promote uptake of the drug as the tumor cell would mistake the ELP-drug complex as being a harmless substance. [1] [9]

Metal recovery

A recent study highlights the first report of thermo-responsive rare-earth elements (REE)-selective protein. The ELP and the REE-binding domain are genetically fused to form REE-selective and thermo-responsive genetically encoded ELP called RELP for the selective extraction and recovery of total REEs. RELP shows a selective and repeatable biosorption platform for REE recovery. The authors highlighted that technology can be adapted to recover other precious metals and commodities. [14]


Related Research Articles

<span class="mw-page-title-main">Alpha helix</span> Type of secondary structure of proteins

An alpha helix is a sequence of amino acids in a protein that are twisted into a coil.

<span class="mw-page-title-main">Biopolymer</span> Polymer produced by a living organism

Biopolymers are natural polymers produced by the cells of living organisms. Like other polymers, biopolymers consist of monomeric units that are covalently bonded in chains to form larger molecules. There are three main classes of biopolymers, classified according to the monomers used and the structure of the biopolymer formed: polynucleotides, polypeptides, and polysaccharides. The Polynucleotides, RNA and DNA, are long polymers of nucleotides. Polypeptides include proteins and shorter polymers of amino acids; some major examples include collagen, actin, and fibrin. Polysaccharides are linear or branched chains of sugar carbohydrates; examples include starch, cellulose, and alginate. Other examples of biopolymers include natural rubbers, suberin and lignin, cutin and cutan, melanin, and polyhydroxyalkanoates (PHAs).

<span class="mw-page-title-main">Protein</span> Biomolecule consisting of chains of amino acid residues

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, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity.

<span class="mw-page-title-main">Protein primary structure</span> Linear sequence of amino acids in a peptide or protein

Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthesis is most commonly performed by ribosomes in cells. Peptides can also be synthesized in the laboratory. Protein primary structures can be directly sequenced, or inferred from DNA sequences.

<span class="mw-page-title-main">Protein folding</span> Change of a linear protein chain to a 3D structure

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

<span class="mw-page-title-main">Polymer backbone</span> Longest chain of covalently-bonded atoms in a polymer

In polymer science, the polymer chain or simply backbone of a polymer is the main chain of a polymer. Polymers are often classified according to the elements in the main chains. The character of the backbone, i.e. its flexibility, determines the properties of the polymer. For example, in polysiloxanes (silicone), the backbone chain is very flexible, which results in a very low glass transition temperature of −123 °C. The polymers with rigid backbones are prone to crystallization in thin films and in solution. Crystallization in its turn affects the optical properties of the polymers, its optical band gap and electronic levels.

<span class="mw-page-title-main">Biomolecule</span> Molecule produced by a living organism

A biomolecule or biological molecule is loosely defined as a molecule produced by a living organism and essential to one or more typically biological processes. Biomolecules include large macromolecules such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as vitamins and hormones. A more general name for this class of material is biological materials. Biomolecules are an important element of living organisms, those biomolecules are often endogenous, produced within the organism but organisms usually need exogenous biomolecules, for example certain nutrients, to survive.

<span class="mw-page-title-main">Elastin</span> Protein allowing tissue in the body to resume shape after stretching

Elastin is a protein that in humans is encoded by the ELN gene. Elastin is a key component of the extracellular matrix in gnathostomes. It is highly elastic and present in connective tissue allowing many tissues in the body to resume their shape after stretching or contracting. Elastin helps skin to return to its original position when it is poked or pinched. Elastin is also an important load-bearing tissue in the bodies of vertebrates and used in places where mechanical energy is required to be stored.

<span class="mw-page-title-main">Protein structure</span> Three-dimensional arrangement of atoms in an amino acid-chain molecule

Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers – specifically polypeptides – formed from sequences of amino acids, which are the monomers of the polymer. A single amino acid monomer may also be called a residue, which indicates a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in which the amino acids lose one water molecule per reaction in order to attach to one another with a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions, such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, cryo-electron microscopy (cryo-EM) and dual polarisation interferometry, to determine the structure of proteins.

<span class="mw-page-title-main">Protein splicing</span> The post-translational removal of peptide sequences from within a protein sequence

Protein splicing is an intramolecular reaction of a particular protein in which an internal protein segment is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins on both sides. The splicing junction of the precursor protein is mainly a cysteine or a serine, which are amino acids containing a nucleophilic side chain. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP). Normally, splicing is associated only with pre-mRNA splicing. This precursor protein contains three segments—an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

<span class="mw-page-title-main">Leucine zipper</span> DNA-binding structural motif

A leucine zipper is a common three-dimensional structural motif in proteins. They were first described by Landschulz and collaborators in 1988 when they found that an enhancer binding protein had a very characteristic 30-amino acid segment and the display of these amino acid sequences on an idealized alpha helix revealed a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The polypeptide segments containing these periodic arrays of leucine residues were proposed to exist in an alpha-helical conformation and the leucine side chains from one alpha helix interdigitate with those from the alpha helix of a second polypeptide, facilitating dimerization.

<span class="mw-page-title-main">Resilin</span> Insect protein

Resilin is an elastomeric protein found in many insects and other arthropods. It provides soft rubber-elasticity to mechanically active organs and tissue; for example, it enables insects of many species to jump or pivot their wings efficiently. Resilin was first discovered by Torkel Weis-Fogh in locust wing-hinges.

<span class="mw-page-title-main">Hydrophobin</span>

Hydrophobins are a group of small cysteine-rich proteins that were discovered in filamentous fungi that are lichenized or not. Later similar proteins were also found in Bacteria. Hydrophobins are known for their ability to form a hydrophobic (water-repellent) coating on the surface of an object. They were first discovered and separated in Schizophyllum commune in 1991. Based on differences in hydropathy patterns and biophysical properties, they can be divided into two categories: class I and class II. Hydrophobins can self-assemble into a monolayer on hydrophilic:hydrophobic interfaces such as a water:air interface. Class I monolayer contains the same core structure as amyloid fibrils, and is positive to Congo red and thioflavin T. The monolayer formed by class I hydrophobins has a highly ordered structure, and can only be dissociated by concentrated trifluoroacetate or formic acid. Monolayer assembly involves large structural rearrangements with respect to the monomer.

<span class="mw-page-title-main">Protein aggregation</span> Accumulation of clumps of misfolded or disordered proteins

In molecular biology, protein aggregation is a phenomenon in which intrinsically-disordered or mis-folded proteins aggregate either intra- or extracellularly. Protein aggregates have been implicated in a wide variety of diseases known as amyloidoses, including ALS, Alzheimer's, Parkinson's and prion disease.

<span class="mw-page-title-main">Peptide amphiphile</span>

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<span class="mw-page-title-main">Abductin</span>

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<span class="mw-page-title-main">HUH-tag</span>

HUH endonucleases (HUH-tags) are sequence-specific single-stranded DNA (ssDNA) binding proteins originating from numerous species of bacteria and viruses. Viral HUH endonucleases are involved in initiating rolling circle replication while ones of bacterial origin initiate bacterial conjugation. In biotechnology, they can be used to create protein-DNA linkages, akin to other methods such as SNAP-tag. In doing so, they create a 5' covalent bond between the ssDNA and the protein. HUH endonucleases can be fused with other proteins or used as protein tags.

<span class="mw-page-title-main">Dextran drug delivery systems</span> Polymeric drug carrier

Dextran drug delivery systems involve the use of the natural glucose polymer dextran in applications as a prodrug, nanoparticle, microsphere, micelle, and hydrogel drug carrier in the field of targeted and controlled drug delivery. According to several in vitro and animal research studies, dextran carriers reduce off-site toxicity and improve local drug concentration at the target tissue site. This technology has significant implications as a potential strategy for delivering therapeutics to treat cancer, cardiovascular diseases, pulmonary diseases, bone diseases, liver diseases, colonic diseases, infections, and HIV.

<i>O</i>-Octadecylhydroxylamine Chemical compound

O-Octadecylhydroxylamine (ODHA) is a white solid organic compound with the formula C18H39NO. ODHA is a noncanonical lipid, which contains a saturated alkyl tail and an aminooxy headgroup. This noncanonical lipid can be site selectively appended to the N-terminal of desired biopolymers such as peptides. ODHA drives the supramolecular assembly of modified protein, presumably through the hydrophobic collapse of ODHA chains.

Ashutosh Chilkoti is an Indian American biomedical engineer, academic and researcher. He is the Alan L. Kaganov Professor of Biomedical Engineering and Senior Associate Dean in the Pratt School of Engineering at Duke University.

References

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  11. ↑ Sun, F; Zhang, WB; Mahdavi, A; Arnold, FH; Tirrell, D (2014). "Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry". PNAS. 111 (31): 11269–11274. Bibcode:2014PNAS..11111269S. doi: 10.1073/pnas.1401291111 . PMC   4128157 . PMID   25049400.
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  13. ↑ Rodríguez-Cabello, José Carlos; Arias, Francisco Javier; Rodrigo, Matilde Alonso; Girotti, Alessandra (February 2016). "Elastin-like polypeptides in drug delivery". Advanced Drug Delivery Reviews. 97: 85–100. doi:10.1016/j.addr.2015.12.007. PMID   26705126.
  14. ↑ Hussain, Zohaib; Kim, Seoungkyun; Cho, Jinhwan; Sim, Gyudae; Park, Youngjune; Kwon, Inchan (2021). "Repeated Recovery of Rare Earth Elements Using a Highly Selective and Thermo-Responsive Genetically Encoded Polypeptide". Advanced Functional Materials. 32 (13): 2109158. doi:10.1002/adfm.202109158. ISSN   1616-3028. S2CID   245519589.
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