Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers

Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val–Pro–Gly–Xaa–Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (T_t), ELPs are soluble, but insoluble when the temperature exceeds T_t. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.     

Effect of detergents on the thermal behavior of elastin‐like polypeptides

Elastin‐like polypeptide (ELP) fusions have been designed to allow large‐scale, nonchromatographic purification of many soluble proteins by using the inverse transition cycling (ITC) method; however, the sensitivity of the aqueous lower critical solubility phase transition temperature (T_t) of ELPs to the addition of cosolutes, including detergents, may be a potential hindrance in purification of proteins with surface hydrophobicity in such a manner. To identify detergents that are known to solubilize such proteins (e.g., membrane proteins) and that have little effect on the T_t of the ELP, we screened a number of detergents with respect to their effects on the T_t and secondary structures of a model ELP (denoted here as ELP180). We found that mild detergents (e.g., n‐dodecyl‐β‐D ‐maltoside, Triton‐X100, and 3‐[(3‐cholamidopropyl) dimethylamino]‐1‐propanesulfonate) do not alter the phase transition behavior or structure (as probed by circular dichroism) of ELP180. This result is in contrast to previous studies that showed a strong effect of other detergents (e.g., sodium dodecylsulfate) on the T_t of ELPs. Our results clearly indicate that mild detergents do not preclude ITC‐based separation of ELPs, and thus that ELP fusions may prove to be useful in the purification of detergent‐solubilized recombinant hydrophobic proteins, including membrane proteins, which are otherwise notoriously difficult to extract and purify by conventional separation methods (e.g., chromatography). © 2012 Wiley Periodicals, Inc.     

Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged,biologically active fusion proteins (ELP-FPs)

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (T_t). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.     

Elastin‐like polypeptide switches: A design strategy to detect multimeric proteins

Elastin‐Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell‐surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a “chemical inducer of dimerization” small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin‐ELPs associate with streptavidin into multimeric complexes with an apparent K_d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates.     

Development of the efficient preparation method for thermoresponsive elastin-like peptides using liquid-phase synthesis combined with fragment condensation strategy

Elastin-like peptides (ELPs) are synthetic peptides that mimic the characteristic hydrophobic amino acid repeat sequences of elastin and exhibit temperature-dependent reversible self-assembly properties. ELPs are expected to be used as temperature-responsive biomolecular materials across diverse industrial and research fields, and there is a requirement for a straightforward method to mass-produce them. Previously, we demonstrated that phenylalanine-containing ELP analogs, namely, (FPGVG)_n, can undergo coacervation with short chains (n = 5). The Fmoc solid-phase peptide synthesis method is one strategy used to synthesize these short ELPs. However, owing to its low reaction efficiency, an efficient method for preparing ELPs is required. In this study, efficient preparation of ELPs was investigated using a liquid-phase synthesis method with a hydrophobic benzyl alcohol support (HBA-tag). Because HBA-tags are highly hydrophobic, they can be easily precipitated by the addition of poor solvents and recovered by filtration. This property allows the method to combine the advantages of the simplicity of solid-phase methods and the high reaction efficiency of liquid-phase methods. By utilizing liquid-phase fragment condensation with HBA-tags, short ELPs were successfully obtained in high yield and purity. Finally, the temperature-dependent response of the ELPs generated through fragment condensation was assessed using turbidity measurements, which revealed a reversible phase transition. Consequently, the ELPs exhibited a reversible phase transition, indicating successful synthesis of ELPs via fragment preparation with tags. These findings provide evidence of the potential for mass production of ELPs using this approach.     

Branched short elastin-like peptides with temperature responsiveness obtained by EDTA-mediated multimerization

Elastin-like peptides (ELPs) exhibit a reversible phase transition, known as coacervation, triggered by temperature changes. This property makes them useful as stimuli-responsive molecular materials for various applications. Among ELPs, short peptide chain lengths have some advantages over long peptide chain lengths because short ELPs can be easily obtained by chemical synthesis, allowing the use of various amino acids, including D-type and unnatural amino acids, at any position in the sequence. Moreover, the incorporated amino acids readily affect the temperature-responsive behavior of ELPs. However, to be utilized in various applications, it is necessary to develop short ELPs and to investigate their temperature-responsive properties. To obtain further insights into the temperature-responsive behavior of the short ELPs, we investigated branched short ELP analogs composed of (FPGVG)_n chains (n = 1 or 2, abbreviated as F1 and F2, respectively). We synthesized multimers composed of four F1 chains or two to four F2 chains using ethylenediaminetetraacetic acid (EDTA) as a central component of multimerization. Our results show that the multimers obtained exhibited coacervation in aqueous solutions whereas linear F1 or F2 did not. Furthermore, the structural features of the obtained multimers were the same as those of linear (FPGVG)₄. In this study, we demonstrated that molecules capable of coacervation can be obtained by multimerization of F1 or F2. The temperature-responsive molecules obtained using short ELPs make it possible to use them as easy-to-synthesize peptide tags to confer temperature responsiveness to various molecules, which will aid the development of temperature-responsive biomaterials with a wide variety of functions.     

Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides

At a specific temperature, elastin-like polypeptides (ELPs) undergo a sharp solubility transition that can be exploited in a variety of applications in biotechnology and medicine. The temperature of the transition varies with ELP sequence, molecular weight, and concentration. We present a single equation of three parameters that quantitatively predicts the transition temperature as a function of ELP length and concentration for an ELP of a fixed composition. This model should be useful both for the design of new ELP sequences that exhibit a desired transition temperature and for the selection of variables to trigger the phase transition of an ELP for a given application.     

Secretion of elastin-like polypeptides with different transition temperatures by Pichia pastoris

Like natural tropoelastin, polypeptides based on an elastin-like VPGXG repeat have a characteristic inverse temperature response, which leads to coacervate formation above a certain transition temperature and which could be useful for a variety of applications. The key advantage of elastin-like polypeptides (ELPs) over (tropo)elastin is a full control over this temperature response by adjustment of either the amino acid composition or the chain length, according to insights provided by extensive research. Future application of ELPs will require efficient ELP production systems, and in a previous article, we described the successful use of Pichia pastoris for secreted production of an ELP, with an overall yield of ≈ 200 mg L(-1). In this study, we investigated the influence of changed amino acid composition and chain length on the yield of secreted ELP. We have found that both parameters have a distinct impact on the overall yield, with higher yield for shorter and more hydrophilic ELPs. Because yield and transition temperature (Tt) thus appear to be positively correlated, we hypothesize that good solubility of ELP below the Tt promotes the secreted production and coacervate formation above Tt decreases it.     

Effects of short elastin‐like peptides on filamentous particles and their transition behavior

While elastin‐like polypeptides and peptides (ELPs) have been used for various stimulus‐responsive applications, details of their switching remain unclear. We therefore constructed a novel series of filamentous phage particles displaying a high surface density of short ELPs. The surface display of ELPs did not disrupt either particle shape or dimensions, and the resulting ELP‐phage particles were colloidally stable over several weeks. However, in spite of a saturating surface density, macroscopic aggregation of ELP‐phages cannot be triggered in water. To investigate the underlying mechanisms we examined conformational changes in the secondary structure of the phage proteins by circular dichroism and tryptophan fluorescence, which indicate partial protein unfolding in ELP‐phage particles. To gain further insight into the ELP itself, analogous “free” ELP peptides were synthesized and characterized. Circular dichroism of these peptides shows the onset of β‐type conformations with increasing temperature, consistent with the accepted view of the microscopic transition that underlies the inverse phase behavior of ELPs. Increased guest residue hydrophobicity was found to depress the microscopic transition temperature of the peptides, also consistent with a previously proposed intramolecular hydrogen‐bonding mechanism. Importantly, our results indicate that although the nanoscale presentation state can suppress macroscopic aggregation of ELPs, microscopic transitions of the ELP can still occur. Given the growing use of ELPs within supra‐molecular scaffolds, such effects are important design considerations for future applications. Biotechnol. Bioeng. 2013; 110: 1822–1830. © 2013 Wiley Periodicals, Inc.     

Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.     

Biodegradation of elastin-like polypeptide nanoparticles

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin-like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (T(t)). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.     

Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair

Elastin-like polypeptides (ELPs) are artificial polypeptides with unique properties that make them attractive as a biomaterial for tissue-engineered cartilage repair. ELPs are composed of a pentapeptide repeat, Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro), that undergo an inverse temperature phase transition. They are soluble in aqueous solution below their transition temperature (T(t)) but aggregate when the solution temperature is raised above their T(t). This study investigates the rheological behavior of an un-cross-linked ELP, below and above its T(t), and also examines the ability of ELP to promote chondrogenesis in vitro. A thermally responsive ELP with a T(t) of 35 degrees C was synthesized using recombinant DNA techniques. The complex shear modulus of the ELP increased by 3 orders of magnitude as it underwent its inverse temperature phase transition, forming a coacervate, or gel-like, ELP phase. Values for the complex shear moduli of the un-cross-linked ELP coacervate are comparable to those reported previously for collagen, hyaluronan, and cross-linked synthetic hydrogels. Cell culture studies show that chondrocytes cultured in ELP coacervate maintain a rounded morphology and their chondrocytic phenotype, characterized by the synthesis of a significant amount of extracellular matrix composed of sulfated glycosaminoglycans and collagen. These results suggest that ELPs demonstrate great potential for use as in situ forming scaffolds for cartilaginous tissue repair.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016     

Modeling the Early Stages of Phase Separation in Disordered Elastin-like Proteins

Elastin-like proteins (ELPs) are known to undergo liquid-liquid phase separation reversibly above a concentration-dependent transition temperature. Previous studies suggested that, as temperature increases, ELPs experience an increased propensity for type II β-turns. However, how the ELPs behave below the phase transition temperature itself is still elusive. Here, we investigate the importance of β-turn formation during the early stages of ELP self-association. We examined the behavior of two ELPs, a 150-repeat construct that had been investigated previously (ELP[V₅G₃A₂-150] as well as a new 40-repeat construct (ELP40) suitable for nuclear magnetic resonance measurements. Structural analysis of ELP40 reveals a disordered conformation, and chemical shifts throughout the sequence are insensitive to changes in temperature over 20°C. However, a low population of β-turn conformation cannot be ruled out based on chemical shifts alone. To examine the structural consequences of β-turns in ELPs, a series of structural ensembles of ELP[V₅G₃A₂-150] were generated, incorporating differing amounts of β-turn bias throughout the chain. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at β-turn regions. This approach was justified by the observation that buried hydrophobic turns are commonly observed to interact in the Protein Data Bank. After dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of β-turn bias, and the results were compared with analytical ultracentrifugation experiments at various temperatures. We find that the temperature dependence of the sedimentation coefficient $\left(s_{20,\text{w}}^{\text{o}}\right)$ can be reproduced by increasing the β-turn content in the structural ensemble. This analysis allows us to estimate the presence of β-turns and weak associations under experimental conditions. Because disordered proteins frequently exhibit weak biases in secondary structure propensity, these experimentally-driven ensemble calculations may complement existing methods for modeling disordered proteins generally.     

Improved non-chromatographic purification of a recombinant protein by cationic elastin-like polypeptides

This paper reports an improvement in the purification of thioredoxin (Trx) expressed from E. coli by inverse transition cycling (ITC) using cationic elastin-like polypeptides (ELPs). Two ELP libraries having 2% and 5% lysine residues and molecular weights ranging from 4 to 61.1 kDa showed greater salt sensitivity in their inverse transition behavior than purely aliphatic ELPs. Expression yield of Trx-ELP fusions was an unpredictable function of guest residue composition, but reducing the molecular weight of the ELP tag generally increased Trx yield. A cationic 4.3 kDa ELP is the shortest ELP used to purify any protein by ITC to date. A 15.9 kDa ELP with a guest residue composition of K:V:F of 1:7:1 was found to be the optimal cationic tag to purify Trx, as it provided 50% greater Trx yield and only required one-fifth the added NaCl for purification of Trx as compared to previously used aliphatic ELP tags.     

Studies on interaction between poly(L-lysine) and DNA of varied G + C contents

Interaction between polylysine and DNA's of varied G + C contents was studied using thermal denaturation and circular dichroism (CD). For each complex there is one melting band at a lower temperature t_m, corresponding to the helix–coil transition of free base pairs, and another band at a higher temperature t′_m, corresponding to the transition of polylysine-bound base pairs. For free base pairs, with natural DNA's and poly(dA-dT) a linear relation is observed between the t_m and the G + C content of the particular DNA used. This is not true with poly(dG)·poly(dC), which has a t_m about 20°C lower than the extrapolated value for DNA of 100% G + C. For polylysine-bound base pairs, a linear relation is also observed between the t′_m and the G + C content of natural DNA's but neither poly(dA-dT) nor poly(dG)·poly(dC) complexes follow this relationship. The dependence of melting temperature on composition, expressed as dt_m/dX_G·C, where X_G·C is the fraction of G·C pairs, is 60°C for free base pairs and only 21°C for polylysine-bound base pairs. This reduction in compositional dependence of T_m is similar to that observed for pure DNA in high ionic strength. Although the t′_m of polylysine-poly(dA-dT) is 9°C lower than the extrapolated value for 0% G + C in EDTA buffer, it is independent of ionic strength in the medium and is equal to the t_m⁰ extrapolated from the linear plot of t_m against log Na⁺. There is also a noticeable similarity in the CD spectra of polylysine· and polyarginine·DNA complexes, except for complexes with poly(dA-dT). The calculated CD spectrum of polylysine-bound poly(dA-dT) is substantially different from that of polyarginine-bound poly(dA-dT).     

Thermally triggered purification and immobilization of elastin-OPH fusions

A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.