Guest Residue Research Articles

Stabilizing effect of solvent and guest residue amino acids on a model alpha-helix peptide

α-Helical peptides of the form For-AAAAAAXAAAAAA-NH2, where X is one of 21 amino acids, have been optimized by use of density-functional theory with the inclusion of solvent by a polarizable continuum model and analyzed by use of the quantum theory of atoms in molecules. Inclusion of solvent results in the transition from a partial 310-helix geometry that was previously observed in gas-phase optimized structures to a fully α-helical geometry, resulting in a substantial loss of NH⋯O i+3 contacts and concurrent formation of NH⋯O i+4 contacts. An increase in the number of N⋯O i+3 contacts was also observed. The total electron density (∑ρ(rc)) at the hydrogen bond critical points (HBCPs) within the peptide backbone increased by up to 160% in the solvated structure. No correlation was found between the ∑ρ(rc) at HBCPs and an α-helix propensity scale, however, the ∑ρ(rc) at HBCPs for the substituted amino acids yields a helix-stabilizing order for the amino acids: Thr>Asn>Ser>Glu>Trp>Arg>Asp>Leu>Cys>His+>Gln>Lys>Met>Ile>Val>Phe>His>Ala>Tyr>Pro>Gly.

Proline Kink Angle Distributions for GWALP23 in Lipid Bilayers of Different Thicknesses

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.

Tryptophan Stabilizes His–Heme Loops in the Denatured State Only When It Is Near a Loop End

We use a host-guest approach to evaluate the effect of Trp guest residues relative to Ala on the kinetics and thermodynamics of formation of His-heme loops in the denatured state of iso-1-cytochrome c at 1.5, 3.0, and 6.0 M guanidine hydrochloride (GdnHCl). Trp guest residues are inserted into an alanine-rich segment placed after a unique His near the N-terminus of iso-1-cytochrome c. Trp guest residues are either 4 or 10 residues from the His end of the 28-residue loop. We find the guest Trp stabilizes the His-heme loop at all GdnHCl concentrations when it is the 4th, but not the 10th, residue from the His end of the loop. Thus, residues near loop ends are most important in developing topological constraints in the denatured state that affect protein folding. In 1.5 M GdnHCl, the loop stabilization is ~0.7 kcal/mol, providing a thermodynamic rationale for the observation that Trp often mediates residual structure in the denatured state. Measurement of loop breakage rate constants, k(b,His), indicates that loop stabilization by the Trp guest residues occurs completely after the transition state for loop formation in 6.0 M GdnHCl. Under poorer solvent conditions, approximately half of the stabilization of the loop develops in the transition state, consistent with contacts in the denatured state being energetically downhill and providing evidence for funneling even near the rim of the folding funnel.

Response of GWALP Transmembrane Peptides to Titration of a Buried Lysine

Designed α-helical peptides such as GWALP23 serve as useful models for probing the influence of polar amino acids within a core transmembrane helical sequence. We incorporated lysine as a guest residue into the membrane-spanning host peptides GWALP23 and closely related Y5GWALP23 (acetyl-GGAL(W5/Y5)LALALAL12AL14ALALW19LAGA-amide). Lysine was introduced at either position 12 or 14 of the host sequences, for which position 12 corresponds to the center. Solid-state NMR spectra of 2H-Ala residues in peptides incorporated into oriented lipid bilayer samples reveal that L14K mutant peptides adopt well-defined orientations in DOPC, DMPC and DLPC. In each lipid membrane, the L14K substitution increases the helix tilt at neutral pH. The L12K substitution, on the other hand, reduces the 2H NMR spectral quality at neutral pH, particularly in the thicker DOPC, suggesting a lack of distinct orientation, as the system struggles to insert a charged lysine into the thicker bilayers. As the positively charged K12 amino group is titrated to higher pH values, nevertheless, the 2H NMR spectral quality improves in DOPC, and the K12 peptides adopt an average orientation nearly matching the one found for both host peptides GWALP23 and Y5GWALP23 (with L12). In similar fashion, titration of K14, in any of the tested lipid bilayer membranes, results in a smaller helix tilt, again much closer to that observed for the L14 peptides without a polar guest residue. Steady-state fluorescence measurements using the Y5GWALP23 series of peptides reveal spectral narrowing and modest blue shifts in Δmax from the W19 reporter, when either K12 or K14 is rendered non-ionized, suggesting a somewhat more hydrophobic environment for the Trp indole ring when the guest Lys side chain is neutral.

On the Combined Analysis of 2H and 15N/1H Solid-State NMR Data for Determination of Transmembrane Peptide Orientation and Dynamics

The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2H-alanine quadrupolar splittings together with 15N/1H dipolar couplings and 15N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA)6LWLAGA-amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5°–35° in lipid bilayer membranes. By comparing individual and combined analyses of specifically 2H- or 15N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<∼10°) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional 15N as well as 2H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with 2H or 15N data alone. Importantly, for peptides that tilt significantly more than 10° from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter.

PPII Propensity of Multiple-Guest Amino Acids in a Proline-Rich Environment

There has been considerable debate about the intrinsic PPII propensity of amino acid residues in denatured polypeptides. Experimentally, this scale is based on the behavior of guest amino acid residues placed in the middle of proline-based hosts. We have used classical molecular dynamics simulations combined with replica-exchange methods to carry out a comprehensive analysis of the conformational equilibria of proline-based host oligopeptides with multiple guest amino acids including alanine, glutamine, valine, and asparagine. The tracked structural characteristics include the secondary structural motifs based on the Ramachandran angles and the cis/trans isomerization of the prolyl bonds. In agreement with our recent study of single amino acid guests, we did not observe an intrinsic PPII propensity in any of the guest amino acids in a multiple-guest setting. Instead, the experimental results can be explained in terms of (i) the steric restrictions imposed on the C-terminal guest amino acid that is immediately followed by a proline residue and (ii) an increase in the trans content of the prolyl bonds due to the presence of guest residues. In terms of the latter, we found that the more guests added to the system, the larger the increase in the trans content of the prolyl bonds, which results in an effective increase in the PPII content of the peptide.

A Proline Kink in GWALP23

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide) is a proven model membrane-spanning peptide (see JACS 130, 12584) that moves “beyond” WALP family peptides by employing, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central alpha-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thickness (see JBC 285, 31723), we utilize GWALP23 as a framework for introducing guest residues within the transmembrane sequence. For example, we have incorporated a central proline residue to give acetyl-GGALW5LALALAP12ALALALW19LAGA-ethanolamide. We have synthesized the resulting GWALP23-P12 with selected 2H and 15N labels for solid-state NMR spectroscopy, to enable analysis of the peptide orientation and segmental tilt in oriented lipid bilayer membranes using combined (2H)-GALA and (15N/1H)-PISEMA methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to proline are tilted substantially with respect to the bilayer normal, by about 34°-40° and 27°-29° (± 6°), respectively, with a proline-induced kink angle of 20°-23°. The proline places restrictions on the dynamics of both segments. As has been described previously for GWALP23, the C-terminal helix ends before Ala-21.

Cucurbituril Complexes of Redox Active Guests

This review summarizes recent work on the chemistry of inclusion complexes formed between redox active guests and cucurbit [n]uril hosts. The large majority of the surveyed research concerns the hosts cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8), while the guests are 4,4-bipyridinium (viologen) and ferrocene derivatives. The work includes dendronized guests, in which a guest residue is attached to a dendron in such a way that host-guest binding interactions are still possible. The main focus of the review is on the electrochemical behavior of the included guests. In general terms complexation by CB7 has a more pronounced effect on the ferrocene derivatives, giving rise to a measurable attenuation of the electrochemical kinetics. In contrast to this, the electrochemical kinetics of the viologen derivatives remains fast (in the voltammetric time scale, with scan rates up to 2 V s-1) upon inclusion complexation by CB7. The larger cavity host CB8 is extremely effective at mediating the redox-driven self-assembly of dendronized viologens. Keywords: Host-guest complexes, electrochemistry, cucurbituril hosts, ferrocene, cobaltocenium, viologen, cucurbit[n]uril (CBn), glycoluril, cyclodextrin, spectroscopic methods, stoichiometry, aromatic protons, pseudorotaxane complex, electrochemical techniques, dialkoxybenzene, voltammograms, cobaltocenium (Cb), ferrocenemethanol, half-wave, dendrimers, metallocene binding

A genetically synthetic protein-based cationic polymer for siRNA delivery

In recent years, a large number of researchers have paid much attention on small interfering RNA (siRNA) after the advent of RNA interference technology, which has been harnessed as an efficient way of sequence-specific gene silencing in gene therapy, enables elucidation of gene functions, and the identification of new drug targets. Despite tremendous progress has been made in novel delivery systems and vectors via formulation of polyplexes and conjugations, such as cationic polymers (LPEI, BPEI), cationic liposome (DOTAP), peptides (CPP), unmet needs still exist. Many cationic agents used for condensing siRNA often exhibits severe cytotoxicity, which limits clinical applications, and is obliged to be handled. Thus great interest in searching for novel and sophisticated polymeric vectors has been spurred. Herein we proposed a genetically synthetic protein-based polymer, which is also referred to as elastin-like polypeptides (ELPs) excerpted from human tropoelastin highly repetitive sequence, Val-Pro-Gly-Xaa-Gly, where the "guest residue" Xaa is any amino acid except Pro. Thus, if we alternate the "guest residue" Xaa to Lys or Arg, to a significant extent, it can emerge as a powerful cationic polymer for siRNA delivery carrier, and hopefully it will be put into practice in the near future.

Formation of host-guest and sandwich complexes by a β-cyclodextrin derivative

A unimer with two host binding sites of β-cyclodextrin and two guest binding sites (two p-tert-butylphenyl moities) has been synthesized. The host and guest residues are linked through an EDTA bridge. Static and dynamic light scattering measurements evidence the formation of supramolecular entities in aqueous solution with a low degree of polymerization (<10). The formation of dendrimer-like structures is demonstrated by TEM measurements. From NMR experiments it is concluded that the p-tert-butylphenyl group is located at three different environments. Two of these sites correspond to the typical locations of a guest in equilibrium with a host, i.e., inside the host cavity and in the bulk solution. The third location corresponds to a p-tert-butylphenyl group sandwiched between the outer surface of at least two cyclodextrin residues and the bridge of the unimer, forming an external complex.

Design of a Soluble Transmembrane Helix for Measurements of Water-Membrane Partitioning

Use of model transmembrane helices and lipid bilayers is a tractable and straightforward approach to obtaining thermodynamic information on fundamental processes of membrane protein folding. The insertion of transmembrane helices from an aqueous phase into membranes, the initial step in the folding process, is especially difficult to investigate because of the insolubility of helices in the aqueous phase. We report here the design of a soluble transmembrane helix, (KR)(5)-AALALAA-AALWLAA-AALALAA-C(NBD)-NH(2) (NBD, 7-nitrobenz-2-oxa-1,3-diazole), consisting of a transmembrane region (AALALAA)(3), a central guest residue (W), and an N-terminal charged tag (KR)(5). Circular dichroism and fluorescence spectroscopy revealed that the peptide dissolved in water as a monomer with the guest residue exposed to the solvent. After the addition of large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the peptide rapidly partitioned into the vesicles and assumed a transmembrane helix. The partitioning Gibbs free energy was estimated to be -34.2 kJ mol(-1) at 25 degrees C. The Trp-to-Gly substitution reduced the partitioning by approximately 1.6 kJ mol(-1). Thus, the transmembrane helix was found to be a useful template for thermodynamic measurements of the partitioning of amino acids from water to the hydrophobic core of membranes.

Hydrogen Bonding of β-Turn Structure Is Stabilized in D2O

The lower critical solution temperature (LCST) of elastin-like polypeptides (ELPs) was investigated as a function of ELP chain length and guest residue chemistry. These measurements were made in both D(2)O and H(2)O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains. Such structural information was obtained by circular dichroism and infrared measurements. Additional thermodynamic data were obtained by differential scanning calorimetry. It was found that there is a greater change in the LCST value between H(2)O and D(2)O for those polypeptides which form the greatest amount of beta-turn/beta-aggregate structure. Moreover, these same molecules were the least hydrophobic ELPs. Therefore, hydrogen bonding rather than hydrophobicity was the key factor in the stabilization of the collapsed state of ELPs in D(2)O compared with H(2)O.

Design of Soluble Transmembrane Helix for Measurement of Water-Membrane Partitioning

Use of model transmembrane helices and lipid bilayers is a tractable and straightforward approach to obtain thermodynamic information on fundamental processes of membrane protein folding. The insertion process of transmembrane helices from aqueous phase to membranes, the initial step of the folding, is especially difficult to investigate because of insolubility of helices in aqueous phase. We report here a design of a soluble transmembrane helix, (KR)5-AALALAA-AALWLAA-AALALAA-C(NBD)-NH2 (NBD, 7-nitrobenz-2-oxa-1,3-diazole), which consists of the transmembrane region (AALALAA)3, the central guest residue (W), and the N-terminal charged tag (KR)5. Circular dichroism and fluorescence spectroscopy revealed that the peptide dissolved into water as a monomer with the guest residue exposed to the solvent. After addition of large unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, the peptides rapidly partitioned into the vesicles and assumed a transmembrane topology. Thus, the designed transmembrane helix was found to be a useful template for thermodynamic measurement of partitioning of amino acids from water to the hydrophobic core of membrane.

The intrinsic conformational propensities of the 20 naturally occurring amino acids and reflection of these propensities in proteins

Here, we compare the distributions of main chain (Phi,Psi) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of Gly-Gly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (Phi,Psi) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation, and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the "intrinsic propensities" of amino acids for particular (Phi,Psi) conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the "random coil" state.

Hydration and conformational mechanics of single, end-tethered elastin-like polypeptides.

We investigated the effect of temperature, ionic strength, solvent polarity, and type of guest residue on the force-extension behavior of single, end-tethered elastin-like polypeptides (ELPs), using single molecule force spectroscopy (SMFS). ELPs are stimulus-responsive polypeptides that contain repeats of the five amino acids Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. We fitted the force-extension data with a freely jointed chain (FJC) model which allowed us to resolve small differences in the effective Kuhn segment length distributions that largely arise from differences in the hydrophobic hydration behavior of ELP. Our results agree qualitatively with predictions from recent molecular dynamics simulations and demonstrate that hydrophobic hydration modulates the molecular elasticity for ELPs. Furthermore, our results show that SMFS, when combined with our approach for data analysis, can be used to study the subtleties of polypeptide-water interactions and thus provides a basis for the study of hydrophobic hydration in intrinsically unstructured biomacromolecules.

Conformational Properties of a Peptide Model for Unfolded α-Helices

Models of protein folding often hypothesize that the first step is local secondary structure formation. The assumption is that unfolded polypeptide chains possess an intrinsic propensity to form these local secondary structures. On the basis of this idea, it is tempting to model the local conformational properties of unfolded proteins using well-established residue secondary structure propensities, in particular, alpha-helix forming propensities. We have used spectroscopic methods to investigate the conformational behavior of a host-guest series of peptides designed to model unfolded alpha-helices. A suitable peptide model for unfolded alpha-helices was determined from studies of the length dependence of the conformational properties of alanine-based peptides. The chosen host peptide possessed a small, detectable, alpha-helix content. Substituting various representative guest residues into the central position of the host peptide at times changed the conformational behavior dramatically, and often in ways that could not be predicted from known alpha-helix forming propensities. The data presented can be used to rationalize some of these propensities. However, it is clear that secondary structure propensities cannot be used to predict the local conformational properties of unfolded proteins.

Intelligent biosynthetic nanobiomaterials for hyperthermic combination chemotherapy and thermal drug targeting of HSP90 inhibitor geldanamycin

An intelligent biosynthetic nanobiomaterial (IBN) platform was explored for drug delivery applications for hyperthermic combination chemotherapy and thermal drug targeting. Geldanamycin (GA), a heat shock protein 90 inhibitor, was conjugated to novel thermosensitive poly(K) 8-poly(VPGXG) 60 block copolymers [K 8-ELP(1-60)] with guest residues as valine, alanine and glycine in a 5:2:3 ratio at the ‘X’ position. The conjugates were completely soluble in PBS and showed a characteristic thermosensitive inverse phase transition. [K 8-ELP(1-60)]-GA conjugate nanoparticles showed a size ranging from 50 to 200 nm depending upon temperature. Relevant to systemic drug delivery in vivo, these IBNs stably disperse in aqueous solution. Cytotoxicity assays have shown that the IBN from [K 8-ELP(1-60)]-GA conjugates exhibits effective hyperthermic combination chemotherapy with facile heat modulation.

Force-Induced Prolyl Cis−Trans Isomerization in Elastin-like Polypeptides

Elastin-like polypeptides (ELPs) are stimulus-responsive polymers that contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. While studying the conformational mechanics of ELPs over a range of solvent conditions by single-molecule force spectroscopy, we noticed that some force-extension curves showed temperature-independent, extensional transitions that could not be fitted with a freely jointed chain or worm-like chain model. Here we show that the observed molecular elongation results from the force-induced peptidyl-prolyl cis-trans isomerization in prolines, which are repeated every fifth residue in the main chain of ELPs. Control experiments with poly(L-proline) demonstrate the similarity of the conformational transition between poly(L-proline) and ELPs. In contrast, the force-extension behavior of poly(L-lysine) showed no deviation in the relevant force range. Force-extension curves in hysteresis experiments showed an elongational difference between extension and relaxation pathways that suggests that the cis conformational state of the prolines could be exhausted on the time scale of the experiment. We present further computational evidence for this mechanism by Monte Carlo simulation of the force-extension behavior using an elastically coupled, two-state model. We believe ours is the first demonstration of force-induced prolyl cis-trans isomerization in proline-containing polypeptides. Our results suggest that single-molecule force spectroscopy could provide an alternate means to assay this important conformational transition in polypeptides.

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