Inverse Transition Cycling Research Articles

Non-chromatographic Method for the Hepatitis B Virus X Protein Using Elastin-Like Polypeptide Fusion Protein

ObjectivesHepatitis B virus (HBV) is a member of the hepadnavirus family. The HBV genome contains four genes designated as S, C, P, and X. The HBV X (HBx) gene encodes for a 16.5-kDa regulatory protein that enhances HBV replication and exerts multifunctional activities. The aim of this study is to describe the rapid and easy purification of HBx using ELP (elastin-like polypeptide) fusion protein. MethodsThe ELP–HBx fusion protein was overexpressed in Escherichia coli. Environmental sensitivity was demonstrated via turbidity and dynamic light scattering as a function of temperature. HBx was purified as an ELP fusion protein. ELPs are biopolymers of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly that undergo an inverse temperature phase transition. ELP follows in temperature and salt consistency, precipitation, and solution repetition (inverse transition cycling) with polypeptide, where it purifies the protein in a simple manner. ResultsFusion proteins underwent supramolecular aggregation at 40 °C in 1 M NaCl and slowly resolubilized at subphysiologic temperatures. ELP domain proteolysis liberated a peptide of comparable size and immunoreactivity to the commercial HBx. ConclusionThis study suggests that HBx can be purified rapidly and easily using inverse transition cycling, and that this method can be applied in determination of HBx 3D structures and HBx stability study.

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.

Expression and non-chromatographic purification of 1,3-propanediol oxidoreductase in Escherichia coli

The gene dha T from Klebsiella pneumoniae encodes 1,3-propanediol oxidoreductase (PDOR). Thermally responsive elastin-like polypeptides (ELPs) was used as a fusion tag to purify the proteins (PDOR). The ELP gene was attached to dha T and ligated into the pET-22b vector. Different NaCl concentrations were employed to decrease the transition temperature (T t ) which was diminished as salt concentration increased. The optimal final concentration of NaCl was 1 M and the corresponding T t was 39.5oC. Enzymatic assays were determined via every step for purification of fusion PDOR. PDOR showed good stability during the purification process, the specific activity in the first and second round of inverse transition cycling (ITC) was 276.1 ± 13.3 and 213.3 ± 10.8 U/mg, respectively. The ELPs fusion PDOR was superior to histidine tagged PDOR in both yield and activity after the purification.

Membrane-Based Inverse Transition Cycling: An Improved Means for Purifying Plant-Derived Recombinant Protein-Elastin-Like Polypeptide Fusions

Elastin-like peptide (ELP) was fused to two different avian flu H5N1 antigens and expressed in transgenic tobacco plants. The presence of the ELP tag enhanced the accumulation of the heterologous proteins in the tobacco leaves. An effective membrane-based Inverse Transition Cycling was developed to recover the ELPylated antigens and antibodies from plant material. The functionality of both the ELPylated neuraminidase and an ELPylated nanobody was demonstrated.

Relevant Elements of a Maize γ-Zein Domain Involved in Protein Body Biogenesis

The N-terminal proline-rich domain of γ-zein (Zera) plays an important role in protein body (PB) formation not only in the original host (maize seeds) but in a broad spectrum of eukaryotic cells. However, the elements within the Zera sequence that are involved in the biogenesis of PBs have not been clearly identified. Here, we focused on amino acid sequence motifs that could be involved in Zera oligomerization, leading to PB-like structures in Nicotiana benthamiana leaves. By using fusions of Zera with fluorescent proteins, we found that the lack of the repeat region (PPPVHL)(8) of Zera resulted in the secretion of the fusion protein but that this repeat by itself did not form PBs. Although the repeat region containing eight units was the most efficient for Zera self-assembly, shorter repeats of 4-6 units still formed small multimers. Based on site-directed mutagenesis of Zera cysteine residues and analysis of multimer formation, we conclude that the two N-terminal Cys residues of Zera (Cys(7) and Cys(9)) are critical for oligomerization. Immunoelectron microscopy and confocal studies on PB development over time revealed that early, small, Zera-derived oligomers were sequestered in buds along the rough ER and that the mature size of the PBs could be attained by both cross-linking of preformed multimers and the incorporation of new chains of Zera fusions synthesized by active membrane-bound ribosomes. Based on these results and on the behavior of the Zera structure determined by molecular dynamics simulation studies, we propose a model of Zera-induced PB biogenesis.

Elastin-like polypeptides as a purification tag for recombinant proteins.

This unit presents a recombinant protein purification method that employs an elastin-like polypeptide (ELP) as a purification tag. ELPs undergo a sharp and reversible phase transition when heated above their lower critical solution temperature. ELPs retain this behavior when they are fused to a protein, and thereby provide a simple method to isolate a recombinant ELP fusion protein from cell contaminants by cycling the solution through the insoluble and soluble phase of the ELP fusion protein using a procedure that is termed Inverse Transition Cycling. This method does not require the use of chromatography, so it is cost-effective, easy to scale up, and easy to multiplex.

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.

Expression and immunogenicity of the mycobacterial Ag85B/ESAT-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy.

This study explored a novel system combining plant-based production and the elastin-like peptide (ELP) fusion strategy to produce vaccinal antigens against tuberculosis. Transgenic tobacco plants expressing the mycobacterial antigens Ag85B and ESAT-6 fused to ELP (TBAg-ELP) were generated. Purified TBAg-ELP was obtained by the highly efficient, cost-effective, inverse transition cycling (ICT) method and tested in mice. Furthermore, safety and immunogenicity of the crude tobacco leaf extracts were assessed in piglets. Antibodies recognizing mycobacterial antigens were produced in mice and piglets. A T-cell immune response able to recognize the native mycobacterial antigens was detected in mice. These findings showed that the native Ag85B and ESAT-6 mycobacterial B- and T-cell epitopes were conserved in the plant-expressed TBAg-ELP. This study presents the first results of an efficient plant-expression system, relying on the elastin-like peptide fusion strategy, to produce a safe and immunogenic mycobacterial Ag85B-ESAT-6 fusion protein as a potential vaccine candidate against tuberculosis.

Influence of elastin‐like peptide fusions on the quantity and quality of a tobacco‐derived human immunodeficiency virus‐neutralizing antibody

The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.

Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELP-protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.

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.

Development of a thermally responsive peptide for sustained deliver of solyble TNF receptor II to attenuate inflammatory events associated with radiculopathys

Background: Tumor necrosis factor alpha (TNFα) is a cytokine that may mediate inflammatory histopathology of the dorsal root ganglion following lumbar disc herniation.1 Soluble TNF receptor II (sTNFRII) competitively binds TNFa with clinical value for painful radiculopathy.2 Bioactive peptides expressed with elastin-like polypeptides (ELP) fusion partners gain a thermally responsive domain, by which they can undergo hydrophobic collapse and separate from solution to aggregate at physiological temperatures.3 Protein release from such a depot may locally sustain drug presence, an effect demonstrated for non-fusion ELP after intra-articular injection.4 
 Methods: We expressed sTNFRII fused to ELP to demonstrate potential bidomain functionality.
 Protein Expression. A gene encoding ELP-(VPGVG)60 was subcloned adjacent to the sTNFRII and transformed into E.coli for expression.5 Protein Safety. Endotoxin content of purified fusion protein was evaluated using a limulus amebocyte lysate endpoint assay and compared to non-fusion ELP using a two-tailed Student’s t-test. Thermal Responsiveness. Dynamic light scattering evaluated the inverse thermal phase transition behaviour of ELP-sTNFRII, and absorbance spectrophotometry quantified the in vitro depot release at 37°C. Fusion Domain Function. Anti-TNFα bioactivity was assessed by the in vitro inhibition of TNFα-induced glutamate production by microglia. Single-factor ANOVA analyzed treatment differences for ELP-sTNFRII, commercial sTNFRII (positive control), and non-fusion ELP (negative control).
 A 44 kDa recombinant fusion protein was expressed from E. coli and purified by inverse transition cycling. 
 Results: Measured endotoxin content for ELP-sTNFRII was comparable to ELP alone (p < 0.01), well below FDA levels for biomedical implants. The fusion protein underwent a thermally-induced phase transition and formed observable aggregates of ~240 nm upon heating to physiological temperatures (Tt = 32°C). Slow release was observed from this depot with a time constant of 21 ± 3 hours. The fusion protein demonstrated anti-TNFα activity in vitro by attenuating TNFα-induced microglial glutamate production, albeit requiring a greater concentration than the free antagonist to achieve the same effect.(p < 0.01). 
 Conclusion: Fusion of a sTNFRII protein to an ELP can serve to generate a thermally-induced drug depot that may sustain anti-cytokine activity of agents delivered locally to a nerve region. Further directions may involve studying in vivo biodistribution after perineural delivery of ELP and in vivo disease modifying activity of this agent.

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.

Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling

A crucial and challenging problem in proteomics is purification, identification, and characterization of proteins, some of which are expressed at very low levels. The preferred method for purification of low abundance proteins exploits multiple affinity purification tags on a single recombinant protein e.g. tandem affinity purification (TAP) [1]. However TAP is both experimentally lengthy, involving many sequential binding, washing and elution steps and costly, requiring two different and expensive resins to recover the purified recombinant protein. Hence processing large amounts of cell lysate makes it prohibitively expensive especially for scale-up. Here we present a new and simple strategy to purify soluble recombinant proteins from E. coli at a protein concentration that approaches the limit of a single protein molecule per cell. This method utilizes the unique aggregation properties of elastin-like polypeptides (ELPs) to capture recombinant fusion proteins composed of a target protein and an ELP tag from cell lysate. ELPs are artificial, genetically encodable polypeptides composed the repeating pentapeptides sequence VPGXG, where the guest residue (X) can be any naturally occurring amino acid except Pro [2,3]. ELPs exhibit a unique reversible inverse phase transition behavior; below a critical transition temperature (Tt) ELPs are highly soluble in aqueous solution, however at temperatures even a few degrees Celsius above Tt, ELP will undergo a solubilityinsolubility phase transition, leading to aggregation of the polypeptide [4]. The Tt of an ELP is a function of a number of variables including identity and stoichiometry of the guest residue, molecular weight, ELP and salt concentration in aqueous solution [5–9]. The environmental sensitivity and reversible solubility of ELPs are retained when expressed as recombinant fusions with proteins. This feature can be exploited for non-chromatographic purification of recombinant proteins by inverse transition cycling (ITC) [10]. In ITC, an ELP fusion protein is selectively separated from other contaminating biomolecules in cell lysate by the sequential and repeated steps of aggregation, centrifugation, and resolubilization of the fusion protein [10,11]. A number of different proteins have been purified by this method using either centrifugation [11–18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14–17]. Proteins expressed at the level of micrograms per liter of culture are difficult to purify by chromatography because of the losses associated with non-specific and irreversible adsorption of the target protein to chromatography resins as well as the relative increase in contamination from host proteins that are non-specifically adsorbed to the chromatography resin are subsequently eluted with the target protein. We demonstrate a new variant of ITC that solves

Functional expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in transgenic plants: purification via inverse transition cycling

In murine models of Crohn's disease, rheumatoid arthritis and colon cancer, IL-6 (interleukin-6) signalling via the sIL-6R (soluble IL-6 receptor; termed IL-6 trans-signalling) has been shown to promote the pathology associated with these conditions. These detrimental activities can, however, be selectively blocked by soluble forms of the gp130 (glycoprotein 130) receptor. Although sgp130 (soluble gp130) therefore represents a viable therapeutic modality for the treatment of these conditions, the mass manufacture of such biologics is often expensive. The advent of molecular farming has, however, provided an extremely cost-effective strategy for the engineering of recombinant proteins. Here, we describe the expression and production of a biologically active sgp130 variant that is expressed in transgenic tobacco plants as an ELP (elastin-like peptide)-fusion protein (mini-gp130-ELP). Mini-gp130-ELP consists of the first three domains of gp130 (Ig-like domain and cytokine binding module) fused to 100 repeats of ELP. Expression of mini-gp130-ELP did not affect the growth rate or morphology of the transgenic plants, and purification was achieved using inverse transition cycling. This approach led to an overall yield of 141 microg of purified protein per g of fresh leaf weight. The purified mini-gp130-ELP specifically inhibited sIL-6R-mediated trans-signalling as measured by binding to the IL-6-sIL-6R complex and through its ability to block sIL-6R-mediated activation of STAT3 (signal transducer and activator of transcription 3) phosphorylation and proliferation in human hepatoma cells and murine pre-B-cells. Consequently, the present study validates the potential application of molecular farming in transgenic tobacco plants as a strategy for the expression and purification of therapeutically advantageous biologics such as sgp130.

Structural optimization of a “smart” doxorubicin–polypeptide conjugate for thermally targeted delivery to solid tumors

A thermoresponsive, genetically engineered, elastin-like polypeptide (ELP) containing a C-terminal cysteine residue was synthesized and purified by inverse transition cycling (ITC) and conjugated to doxorubicin (Dox) molecules through four different pH-sensitive, maleimide-activated, hydrazone linkers. The efficiency of Dox activation, conjugation ratios to ELP and biophysical characterization–hydrodynamic radius (Rh) and the temperature transition kinetics–of the ELP–Dox conjugates and pH-mediated release of Dox were quantified in this study. Conjugation ratios of the maleimide-activated Dox to the thiol group of a unique cysteine in the ELP were close to unity. The Rh of the conjugate increased as the linker length between the ELP backbone and Dox was increased. The linker structure and length had little effect on the Tt of the ELP–Dox conjugates, as all conjugates exhibited Tt's that were similar to the native ELP. However, the ELP–Dox conjugates with longer linkers exhibited slower transition kinetics compared to the ELP–Dox conjugates with shorter linkers. The highest release of the ELP–Dox conjugate by cleavage of the hydrazone bond at pH 4 was nearly 80% over 72 h and was exhibited by the conjugate with the shortest linker.

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin‐like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.

Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation.

Research on spider silk proteins has led to the possibility of designing genetically engineered silks according to defined material properties. Here we show the efficient and stable production of spider silk-elastin fusion proteins in transgenic tobacco and potato plants by retention in the ER. The proteins were purified by a simple method, using heat treatment and 'inverse transition cycling'. Laboratory scale extraction of 1 kg tobacco leaf material leads to a yield of 80 mg pure recombinant spider silk-elastin protein. As a possible application, as well as to demonstrate biocompatibility, the growth of anchorage-dependent mammalian cells on spider silk-elastin coated culture plates was compared with conventional coatings such as collagen, fibronectin and poly-D-lysine. The anchorage-dependent chondrocytes showed similar growth behaviour and a rounded phenotype on collagen and on spider silk-elastin coated plates and the proliferation was remarkably superior to untreated polystyrene plates.

Fabrication of Stimulus-Responsive Nanopatterned Polymer Brushes by Scanning-Probe Lithography

Stimulus-responsive, surface confined poly(N-isopropylacrylamide) (pNIPAAM) brush nanopatterns were prepared on gold-coated silicon substrates in a “grafting-from” approach that combines “nanoshaving”, a scanning probe lithography method, with surface-initiated polymerization using atom transfer radical polymerization (ATRP). The reversible, stimulus-responsive conformational height change of these nanopatterned polymer brushes was demonstrated by inverse transition cycling in water, and water−methanol mixtures (1:1, v:v). Our findings are consistent with the behavior of laterally confined and covalently attached polymer chains, where chain mobility is restricted largely to the out-of-plane direction. Our nanofabrication approach is generic and can likely be extended to a wide range of vinyl monomers.

Purification of recombinant proteins by fusion with thermally-responsive polypeptides.

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.