Elastin-like Polypeptide Tag Research Articles

Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicagosativa

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/μg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.

Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen.

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.

Separation and Purification of Recombinant β-Glucosidase with Hydrophobicity and Thermally Responsive Property from Cell Lysis Solution by Foam Separation and Further Purification.

The aim of this study was to separate and purify recombinant β-glucosidase (GLEGB) with elastin-like polypeptide (ELP) and graphene-binding peptide (GB) from cell lysis solution by foam separation and further purification. The study of foam property of GLEGB cell lysis solution indicated that it had excellent foaming property and foam stability, which was suitable for foam separation. This could be due to the GB tag with hydrophobicity, which made the recombinant β-glucosidase with GB preferentially adsorb on the surface of bubbles. At optimum operating conditions of foam separation, the enzyme activity recovery of GLEGB could reach 95.63 ± 1.0%. The foam solution of GLEGB was further purified based on the thermally responsive property of the ELP tag, and the purification fold of GLEGB could reach 29.6 ± 0.5 at the optimum operating conditions. The prominent purification effect indicates that this technique is a simple and efficient technique for the separation and purification of recombinant enzymes.

A versatile tag for simple preparation of cutinase towards enhanced biodegradation of polyethylene terephthalate

Enzymatic degradation of polyethylene terephthalate (PET) suffered from challenges such as complex and costly enzyme preparation, difficult access to PET substrates, poor reusability of free enzymes and sometimes MHET inhibitions. Herein, we propose an “all-in-one” strategy to address these issues with a well-designed elastin-like polypeptides (ELPs) tag. The preparation of the ELPs-tagged cutinase (ET-C) was efficient and easy to scale up by centrifugation, with an activity recovery of 57.55 % and a yield of 160 mg/L. Besides, the activity of the ET-C was 1.3 and 1.66-fold higher in degrading PET micro- and macro-plastics compared to wild-type cutinase. The self-immobilized cutinase (ET-C@SiO2) obtained by the ELPs-mediated biosilicification exhibited high loading capacity, activity, and thermostability and maintained 77.65 % of the original activity after 10 reuses. Interestingly, the product of the ET-C was TPA, whereas the wild-type was TPA and MHET. This is a simple way to release the intermediates inhibition compared with the existing methods. Our results demonstrated the feasibility of the versatile ELPs tag, which will pave an alternative economic way for scalable PET biodegradation.

Synthesis of single-chain antibody fragment fused to the elastin-like polypeptide in Nicotiana benthamiana and its application in affinity precipitation of difficult to produce proteins.

Plants are economical and sustainable factories for the production of recombinant proteins. Currently, numerous proteins produced using different plant-based systems with applications as cosmetic and tissue culture ingredients, research and diagnostic reagents, and industrial enzymes are marketed worldwide. In this study, we aimed to demonstrate the usefulness of a plant-based system to synthesize a single-chain antibody (scFv)-elastin-like polypeptide (ELP) fusion to be applied as an affinity precipitation reagent of the difficult to produce recombinant proteins. We used the human tissue transglutaminase (TG2), the main celiac disease autoantigen, as a proof of concept. We cloned a TG2-specific scFv and fused it to a short hydrophobic ELP tag. The anti-TG2-scFv-ELP was produced in Nicotiana benthamiana and was efficiently recovered by an inverse transition cycling procedure improved by coaggregation with bacteria-made free ELP. Finally, the scFv-ELP was used to purify both plant-synthesized human TG2 and also Caco-2-TG2. In conclusion, this study showed for the first time the usefulness of a plant-based expression system to produce an antibody-ELP fusion designed for the purification of low-yield proteins.

Polymer size affects biodistribution and placental accumulation of the drug delivery biopolymer elastin-like polypeptide in a rodent pregnancy model

IntroductionFusion of therapeutic agents to Elastin-like Polypeptide (ELP) is a novel drug delivery strategy for prevention of placental drug transfer. Previous studies have used a 60 kDa ELP tag for this purpose. However, placental transfer of ELP may be size dependent. The goal of this study was to measure the effects of ELP polymer size on pharmacokinetics, biodistribution, and placental transfer of ELP. MethodsThree ELPs ranging from 25 to 86 kDa (4.1–6.8 nm hydrodynamic radius) were fluorescently labeled and administered by i.v. bolus to pregnant Sprague Dawley rats on gestational day 14. Plasma levels were monitored for 4 h, organ levels and placental transfer determined by ex vivo fluorescence imaging, and placental localization determined by confocal microscopy. ResultsIncreasing ELP size resulted in slower plasma clearance and increased deposition in all major maternal organs, except in the kidneys where an opposite effect was observed. Placental levels increased with an increase in size, while in the pups, little to no ELP was detected. DiscussionPharmacokinetics and biodistribution of ELPs during pregnancy are size dependent, but all ELPs tested were too large to traverse the placental barrier. These studies verify that ELP fusion is a powerful method of modulating half-life and preventing placental transfer of cargo molecules. The tunable nature of the ELP sequence makes it ideal for drug delivery applications during pregnancy, where it can be used to target drugs to the mother while preventing fetal drug exposure.

Column Chromatography Free Purification of Recombinant α-Amylase from Bacillus licheniformis by Tagging with Hydrophobic Elastin Like Polypeptide

Thermal sensitive and highly hydrophobic elastin like polypeptide (ELP) tends to undergo inverse transition cycling (ITC) which can be used for non chromatographic purification. The present study reports non-chromatographic purification of industrially important α-amylase tagged with ELP and compared with IMAC (Immobilized metal affinity chromatography) purified α-amylase. amyL-Gene encoding α-amylase from Bacillus licheniformis was cloned and expressed in E. coli BL21. The expressed protein with His-tag was purified through IMAC using Ni–NTA matrix. Three ELP genes encoding repeats of pentapeptide (Val-Pro-Gly-Val-Gly)n with variable length (V = 20, 21, 22) were synthesized through PCR using overlapping primers. To generate ELP tagged α-amylase, amyL was placed at N-terminal of ELPs and transformed to E. coli BL21 for expression. After ITC, ELP22 at 30 °C showed maximum yield. α-Amylase purification through ITC and IMAC showed 2.9 and 1.72-fold purification, respectively. Furthermore, physical parameters of ELP tagged α-amylase have shown improvement with working temperature and thermal stability in comparison to His-tag α-amylase. The k cat /K m for ELP tag and His-tag α-amylase was found to be 61.4 and 23.7 mg−1 min−1, respectively, which shows that ELP-tag increased the enzyme efficiency. In conclusion, the ELP-tag purification strategy can be applied to industrially relevant enzyme for purification by the non-chromatography method.

Enhanced thermal stability of lichenase from Bacillus subtilis 168 by SpyTag/SpyCatcher-mediated spontaneous cyclization.

BackgroundSpyTag is a peptide that can form an irreversible covalent linkage to its 12 kDa partner SpyCatcher via a spontaneous isopeptide bond. Herein, we fused SpyTag at the N-terminal of lichenase and SpyCatcher at C-terminal so that the termini of lichenase were locked together by the covalent interaction between the partners. In addition, an elastin-like polypeptides tag was subsequently attached to the C-terminus of SpyCatcher, thereby facilitating the non-chromatographic purification of cyclized lichenase.ResultsThe study showed that the optimum temperature of the cyclized lichenase was about 5 °C higher in comparison to its linear counterpart. Moreover, nearly 80 % of the cyclized lichenase activities were retained after 100 °C exposure, whereas the linear form lost almost all of its activities. Therefore, the cyclized variant displayed a significantly higher thermal stability as temperature elevated and was resistant to hyperthermal denaturation. Besides, the Km value of the cyclized lichenase (7.58 ± 0.92 mg/mL) was approximately 1.7-fold lower than that of the linear one (12.96 ± 1.93 mg/mL), indicating a higher affinity with substrates.ConclusionsThis new SpyTag/SpyCatcher cyclization strategy is deemed as a generalized reference for enhancing enzyme stability and can be effectively customized to the cyclization of various enzymes, hence a tremendous potential for successful application in the biocatalytic conversion of biomass to produce fuels and chemicals.

Enhancement of the solubility and stability of d-amino acid oxidase by fusion to an elastin like polypeptide

An elastin-like polypeptide (ELP) was fused to d-amino acid oxidases (DAAO). ELP–DAAO exhibited a better solubility in aqueous solutions than DAAO, and its enzymatic activity is about 1.6 times that of DAAO. The stability of the proteins was investigated by interacting with urea at various concentrations. The circular dichroism and fluorescence spectra were measured. The results demonstrated that that ELP–DAAO exhibited a much better stability than DAAO, and ELP–DAAO has retained the α-helix content with a high percentage even at a high urea concentration. The results of this work have demonstrated that the ELP tag can be utilized to purify DAAO, in the meantime the solubility and stability of the enzyme are improved.

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags.

BackgroundPectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity.ResultsPolygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves.ConclusionOur results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from <em>Escherichia coli</em>

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from Escherichia coli.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

BackgroundPlants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum.ResultsThe accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants.ConclusionThe use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

Enhancing cellular uptake of GFP via unfolded supercharged protein tags

One of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics.

High‐level production of human interleukin‐10 fusions in tobacco cell suspension cultures

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

Expression and one-step purification of a β-galactosidase by fusion with elastin-like polypetides

Thermally triggered reversible phase transition of elastin-like polypeptide (ELP) allows for a simple, economical and scalable procedure of protein purification. This technique is especially useful for purifying salt-requiring enzymes such as halophilic enzymes which require high salt concentration to keep natural structure and activity. In this study, a highly hydrophilic/acidic β-galactosidase cloned from halotolerant Planococcus sp.L4 was used as a target protein to apply ELP tags for purification. A high-level expression of β-galactosidase tagged with 80 repeats of Val-Pro-Gly-Val-Gly pentapeptide (galactosidase-ELP[V5-80]) was achieved in Escherichia coli BLR(DE3) at 21°C for 24h, accounting for around 50% of the total protein. The enzyme activity of the fusion by optimized protocol should be reached as much as 3 folds of that by rapid IPTG-induction, implying that measures to avoid possible errors during protein expression can be helpful for keeping bioactivities. The optimal condition for precipitating ELP-tagged protein was performed with a simple, rapid and sensitive method by examining the activity of supernatant after the first-round hot spin. The fusion protein aggregated effectively at 37°C with 1.5M ammonium sulfate and yielded highly pure protein with a recovery higher than 90% by one cycle. These results suggested that inverse transition cycling (ITC) process provides a potential for the large-scale purification of halophilic β-galactosidase.

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

Purification of Escherichia coli RNA polymerase using a self‐cleaving elastin‐like polypeptide tag

A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

Expression and Purification of an Antimicrobial Peptide by Fusion with Elastin-like Polypeptides in Escherichia coli

Different carrier molecules have been fused to antimicrobial polypeptides (AMPs) to facilitate recombinant protein expression and purification. Some of them have improved the stability of AMPs and reduced the toxicity to host cells, but most current strategies still have some problems to be solved such as poor yield, low purity, high expense, time-consumption, and difficulty in scaling-up. Here, we introduced the elastin-like polypeptides (ELPs) as a fusion partner to express an antimicrobial polypeptide halocidin18 (Hal18). By the reversible soluble-insoluble phase transition, 69 mg of the fusion protein were purified from 1 l of culture medium with the purity of nearly 95%. After cleavage with hydroxylamine, the ELP's tag was easily separated from Hal18 in the next round of inverse transition cycle and Hal18 (1.7 mg, approximately 1.9 kDa) was mainly found in the supernatant with a recovery of about 47% and purity of 60%. Antimicrobial activity showed that Hal18 had strong antimicrobial activity against Escherichia coli and Micrococcus luteus but weak activity against Pichia pastoris.

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions.

BackgroundElastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants.ResultsThe effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system.ConclusionAn endoplasmic reticulum-targeted elastin-like polypeptide fusion approach provides an effective strategy for depositing large amounts of concentrated heterologous protein within the limited space of the cell via storage in stable protein bodies. Furthermore, encapsulation of recombinant proteins into physiologically inert organelles can function to insulate the protein from normal cellular mechanisms, thus limiting unnecessary stress to the host cell. Since elastin-like polypeptide is a mammalian-derived protein, this study demonstrates that plant seed-specific factors are not required for the formation of protein bodies in vegetative plant tissues, suggesting that the endoplasmic reticulum possesses an intrinsic ability to form protein body-like accretions in eukaryotic cells when overexpressing particular proteins.