RESUMO
Immunoglobulin M (IgM) antibodies hold promise as anticancer drugs and as agents for promoting immune homeostasis. This promise has not been realized due to low expression levels in mammalian cells producing IgM class antibodies, and the failure of protein A chromatography for IgM purification. Here, we describe a nonchromatographic platform for quantitatively capturing IgMs at neutral pH, which is then recovered with 86%-94% yield and >95% purity at pH 3. The platform contains micelles conjugated with the [(bathophenanthroline)3 :Fe2+ ] amphiphilic complex. Inclusion of amino acid monomers, for example, phenylalanine or tyrosine, during conjugation of detergent micelles, allows subsequent extraction of IgMs at close to neutral pH. With the successful implementation of this purification platform for both polyclonal humans and bovine IgMs, we anticipate similar results for monoclonal IgMs, most relevant for the pharmaceutical industry.
Assuntos
Detergentes , Micelas , Animais , Anticorpos Monoclonais/metabolismo , Bovinos , Humanos , Imunoglobulina M/metabolismo , Mamíferos/metabolismo , Proteína Estafilocócica ARESUMO
In the decades'-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe2+ or Ni2+ were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10-200 µm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.
Assuntos
Bacteriorodopsinas , Micelas , Bacteriorodopsinas/química , Cristalização , Detergentes/química , ÁguaRESUMO
We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose-synthesized, peptide amphiphiles, C10 -(Asp)5 and C10 -(Lys)5 . Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C10 -(Asp)5 are conjugated (a) via a water-soluble, penta-Lys mediator or (b) to micelles containing the C10 -(Lys)5 peptide amphiphile. Both routes lead to phase separation in the form of oil-rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light-driven proton pump King Sejong 1-2 (KS1-2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.
Assuntos
Peptídeos/química , Bombas de Próton/química , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Micelas , Microscopia , ÁguaRESUMO
The dearth of high quality, three dimensional crystals of membrane proteins, suitable for X-ray diffraction analysis, constitutes a serious barrier to progress in structural biology. To address this challenge, we have developed a new crystallization medium that relies on the conjugation of surfactant micelles via base-pairing of complementary hydrophobic nucleosides. Base-pairs formed at the interface between micelles bring them into proximity with each other; and when the conjugated micelles contain a membrane protein, crystal nucleation centers can be stabilized, thereby promoting crystal growth. Accordingly, two hydrophobic nucleoside derivatives - deoxyguanosine (G) and deoxycytidine (C), each covalently bonded to a 10 carbon chain were synthesized and added to an aqueous solution containing octyl ß-d-thioglucopyranoside micelles. These hydrophobic nucleosides induced the formation of oil-rich globules after 2days incubation at 19°C or after a few hours in the presence of ammonium sulfate; however, phase separation was inhibited by 100mM GMP. The presence of the membrane protein bacteriorhodopsin in the conjugated - micellar dispersion resulted in the growth within the colorless globules of a variety of purple crystals, the color indicating a functional protein. On this basis, we suggest that conjugation of micelles via base-pair complementarity may provide significant assistance to the structural determination of integral membrane proteins.
Assuntos
Desoxicitidina/química , Desoxiguanosina/química , Bacteriorodopsinas/química , Pareamento de Bases , Cristalização , Cristalografia por Raios X/métodos , MicelasRESUMO
We describe two alternative and complementary purification methods for halorhodopsin and bacteriorhodopsin. The first relies on a unique form of detergent micelles which we have called engineered-micelles. These are specifically conjugated in the presence of [hydrophobic chelator:Fe(2+)] complexes and form detergent aggregates into which membrane proteins partition, but hydrophilic water-soluble proteins do not. The approach was tested on the membrane protein, bacteriorhodopsin (bR), with five non-ionic detergents (OG, OTG, NG, DM, and DDM), commonly used in purification and crystallization of membrane proteins, in combination with the commercially available bathophenanthroline or with one of the three synthesized phenanthroline derivatives (Phen-C10, Phen-C8 and Phen-C6). Our results show that bR is extracted efficiently (60-86%) and directly from its native membrane into diverse detergent aggregates with preservation of its native conformation, while 90-95% of an artificial contaminating background is excluded. For implementation of the second method, based on engineered-membranes, the use of detergents, which in some cases may produce protein denaturation, is not required at all. Protein-containing membranes are conjugated via the same hydrophobic [chelator:metal ion] complexes but maintain the membrane protein in its native bilayer environment throughout the process. This method is demonstrated on the membrane protein halorhodopsin from Natronomonas pharaonis (phR) and leads to good recovery yields (74-89%) and removal of >85% of artificial background impurities while preserving the native state of phR. The detailed structure of the hydrophobic chelator used has been found to have a marked effect on the purity and yield of both methods.
Assuntos
Bacteriorodopsinas/isolamento & purificação , Detergentes/química , Halorrodopsinas/isolamento & purificação , Engenharia de Proteínas , MicelasRESUMO
Conjugated engineered-micelles, i.e. micelles that are composed of nonionic detergents and hydrophobic chelators and subsequently conjugated in the presence of divalent metal ions, have been shown to be remarkably suited to the task of membrane protein purification, maintaining these proteins in their native state. They also efficiently solubilize highly hydrophobic antibiotics. To date, however, the morphological changes induced in the initially spherical or ellipsoidal micelles by conjugation have not been explored. In this study, the very rapid sample-vitrification protocol of cryogenic transmission electron microscopy (cryo-TEM) has been used to capture structural transformations that engineered-micelles undergo immediately following conjugation with the [(bathophenanthroline)3:Fe(2+)] hydrophobic complex. We found that condensed thread-like aggregates are formed when the detergents used are: octyl ß-D-glucopyranoside (OG), octyl ß-D-thioglucopyranoside (OTG) or pentaethylene glycol monododecyl ether (C12E5). However, with ß-D-maltoside (DM), n-dodecyl ß-D-maltoside (DDM) or ß-D-glucopyranoside (DDG), lamellar structures, some of which appear as stacked lamellae or multilamellar vesicles (MLV's), were observed. Such architectural changes occur under very mild conditions i.e. low detergent concentration, no temperature or pH alterations and without the presence of any precipitants such as PEG or ammonium sulfate.
RESUMO
The SARS-COV-2 virus is a deadly agent of inflammatory respiratory disease. Since 2020, studies have focused on developing new therapies based on galactose-rich IgA antibodies. Clinical surveys have also revealed that galactose-deficient IgA1 polymerizes in serum, producing IgA nephropathy, which is a common cause of kidney failure in young adults. Here we show that IgA1-IgA2 dimers are efficiently and economically purified in solution via conjugated nonionic surfactant micellar aggregates. Quantitative capture at pH 7 and extraction at pH 6.5 can avoid antibody exposure to acidic, potentially denaturing conditions. Brij-O20 aggregates lead to the highest process yields (88-91%) and purity (94%). Recovered IgA dimers preserve their native secondary structure and do not self-associate. Increasing the reaction volume has little impact on yield or purity. By introducing an efficient, inexpensive IgA purification protocol, we assist pharmaceutical firms and research laboratories in developing new IgA-based therapies as well as in increasing our understanding of IgA1 polymerization.
RESUMO
We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (â¼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.
RESUMO
A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, ß-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe(2+) ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 °C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins.
Assuntos
Proteínas de Membrana/isolamento & purificação , Micelas , Bacteriorodopsinas/isolamento & purificação , Microscopia Eletrônica de TransmissãoRESUMO
Immunoglobulin-G (IgG) (â¼150 kDa) antibodies confer longer term immunity against bacterial or viral infections than the heavier IgM's (â¼900 kDa), which are generally detectable in blood circulation in response to more recently acquired infections. There may be, however, a time overlap, which is problematic for diagnostic purposes, in the interests of which it is essential to separate IgM's from IgG's. We describe a purification platform, functioning at pH 6.5, containing Tween-20, or Brij-O20, non-ionic detergent micelles, mixed with the sugar-rich detergent dodecyl maltoside (DDM), amino acid monomer tyrosine (Tyr), and conjugated by the amphiphilic complex [(bathophenanthroline)3: Fe2+]. Using conjugated Brij-O20 micelles, with input molar ratio IgG: IgM 9:1, IgG is recovered at 10 °C with 85-90% yield, (by SDS-PAGE densitometry) and ≥95% purity (also by SDS-PAGE), while IgM's are recovered at lower yields (28-34%) and contain small amounts of co-extracted IgG's. Addition of E. coli lysate as an artificial contamination background does not reduce the yield or purity of the recovered IgG. Tween-20/DDM/Tyr micelles lead to IgG purity ≥95% similar to that of Brij-O20, but with lower process yields (64-70%, by densitometry). Chromatographic separation with Protein A or Protein G resins leads to yields comparable to those obtained with Brij-O20 micelles, but with lower purity.
Assuntos
Imunoglobulina G , Tensoativos , Imunoglobulina M , Polissorbatos , Micelas , Detergentes , Escherichia coli , LipoproteínasRESUMO
Specific conjugation of decyl ß-D-maltoside (DM) or dodecyl ß-D-maltoside (DDM) detergent micelles is accomplished between pH 7.0-8.5 in the presence of an amphiphilic analog of the amino acid histidine, bound to a 10-carbon hydrocarbon chain (His1-C10) and Ni2+ ions. Following addition of 10-15 wt% PEG-6000 as precipitant, phase separation in the form of oil-rich globules (30-600 µm) is observed by light microscopy. Other divalent cations: Zn2+, Fe2+, Cu2+ lead to dark precipitates rather than colorless globules; while Mg2+, Ca2+ do not promote any phase separation at all. Even in the absence of precipitant, dynamic light scattering (DLS) measurements demonstrate that DM micelles (hydrodynamic size ~ 6 nm) or DDM micelles (8 nm) self-associate into larger particles (9 nm and 411 nm for DM; 10 nm and 982 nm for DDM) in the presence of His1-C10 and nickel ions. Micellar conjugation is partially reversible in the presence of water soluble 50 mM EDTA, histidine or imidazole chelators. Cryo-transmission electron microscopy (cryo-TEM) imaging revealed the formation of non-uniformly dense detergent aggregates for both DM and DDM micelles in the presence of precipitant. The possible utility of such His1-tagged DM or DDM micelles for promoting crystallization of integral membrane proteins is discussed.
RESUMO
Industrial scale production of therapeutic monoclonal antibodies (mAbs) is commonly achieved with Protein A chromatography, a process that requires exposure of the antibody to strongly acidic conditions during the eluting step. Exposure to acid inactivates virus contaminants but may, in parallel, lead to antibody aggregation that must be eliminated or kept at acceptably low levels. This report seeks to provide a practical method for overcoming a long-standing problem. We show how Brij-O20 detergent micelles, conjugated by the amphiphilic [(bathophenanthroline)3:Fe2+] complex in the presence of amino acid monomers: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), isoleucine (Ile) or valine (Val), efficiently capture polyclonal human IgG (hIgG) at neutral pH and allow its recovery by extraction either at pH 4 (85-97% yield) or at pH 6.3 (72-84% yield). Of the five amino acid monomers surveyed, Phe or Tyr produced the highest overall process yield at both pH 4 and 6.3. The monomeric state of the purified hIgG's was confirmed by dynamic light scattering (DLS). Potential advantages of the purification method are discussed.
Assuntos
Aminoácidos , Micelas , Detergentes , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G , TirosinaRESUMO
We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates led to high purity of the recovered Ab's (>95 %, by gel densitometry). Good overall Ab recovery yields were observed with Tween-20 (80-83 %), Brij-O20 (85-87 %) and Triton X-100 (87-90 %), while Pluronic F-127 was less efficient (42-53 %). Of additional importance is the finding that the process was performed by filtration rather than centrifugation, thereby allowing a continuous purification mode that led to the recovery of monomeric IgG, as determined by dynamic light scattering and preservation of Ab specificity as measured by ELISA. The amphiphilic chelator, bathophenanthroline (batho) was recycled almost quantitatively (95 %) by crystallization. Good IgG recovery yields of â¼80 % were also observed when Ab concentrations were increased from 1â¯mg/mL to 3-5â¯mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies, are discussed.
Assuntos
Anticorpos/isolamento & purificação , Detergentes/química , Proteína Estafilocócica A/química , Anticorpos/química , Cromatografia , Compostos Ferrosos/química , Ligantes , Micelas , Estrutura Molecular , Fenantrolinas/química , Polissorbatos/químicaRESUMO
The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab')2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab')2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab')2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab')2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab')2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgG's as test samples.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Proteína Estafilocócica A/química , Anticorpos Monoclonais/química , Cromatografia de Afinidade , Escherichia coli/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , MicelasRESUMO
We report the first observation of an efficient, native membrane conjugation mechanism via positively charged, linear oligo-amines. Clustering of membrane fragments relies on electrostatic interactions between the net negative charge of the membranes and the positively charged, water-soluble mediators. This conjugation principle is demonstrated with two different bacterial membranes in which are embedded either the intrinsic membrane protein (MP) bacteriorhodopsin (bR) or the more recently identified xanthorhodopsin (XR). As determined by their characteristic UV-vis absorption spectra and by circular dichroism, the MPs are not significantly perturbed by the oligo-amines carrying from +3 to +6 positive charges. Light microscopy and scanning electron microscope (SEM) imaging provide direct evidence for membrane conjugation. Process efficiency was found to be correlated with the net charge of the oligo-amine used. Membrane conjugation is accomplished within a wide range of pH values (7-2.5); is reversed by NaCl; and does not require the presence of a precipitant (e.g. PEG) nor Ca2+ ions. Some evidence for bilayer fusion is also observed, but only in the presence of the +6 oligo-amine analog.
Assuntos
Aminas/química , Proteínas de Bactérias/química , Bacteriorodopsinas/química , Rodopsinas Microbianas/química , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Eletricidade Estática , Propriedades de SuperfícieRESUMO
A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1-2 (KS1-2), using the amphiphilic 4,4'-dinonyl-2,2'-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal-chelator] complexes leads to purple, hexagonal crystals (50-75 µm in size) of bR or pink, rectangular/square crystals (5-15 µm) of KS1-2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.
Assuntos
Bacteriorodopsinas/química , Cristalização/métodos , Micelas , Quelantes/química , Compostos Ferrosos/química , Níquel/química , Tioglucosídeos/química , Zinco/químicaRESUMO
We have recently introduced a non-chromatographic alternative for antibody (Ab) purification, one which does not require the use of Protein A. With this approach, polyclonal human or mouse immunoglobulins (IgG's) are captured almost quantitatively by Tween-20 micelles conjugated with a [chelator:divalent metal cation] complex. Target IgG structure remains native even following extraction from the surfactant aggregate. In the present work, we explore the effect of varying both components of the [metal:chelator] pair on the yield of purified Ab. Capture efficiency is observed to correlate with the formation of sufficiently large detergent aggregates, as determined by dynamic light scattering (DLS) and polyacrylamide gel electrophoresis (PAGE). This, in turn, depends on the rigidity and aromaticity of the chelator. Detergent aggregates are stable over a wide range of pH values (pHâ¯=â¯3-9). Under acidic conditions (3-3.8) they lead to good IgG recovery yields (70-78%) with purity similar to that obtained with Protein A chromatography while maintaining the monomeric state of the IgG's. Finally, the influence of the environment and the presence of various water-soluble chelators (e.g. EDTA, histidine, imidazole) on process efficiency, is described.
Assuntos
Quelantes/química , Imunoglobulina G/isolamento & purificação , Metais/química , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Micelas , Polissorbatos , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismoRESUMO
We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated "Engineered-micelles" built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe2+ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70-80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.
Assuntos
Imunoglobulina G/isolamento & purificação , Micelas , Animais , Humanos , Imunoglobulina G/química , Camundongos , Soroalbumina Bovina/químicaRESUMO
A general approach for anti-hapten antibody purification utilizing double-modified albumins is presented. Purification is based on simultaneous modification of an albumin with a hapten (e.g. fluorescein) and desthiobiotin. Three distinct albumins (BSA, HSA and ovalbumin) were modified accordingly and evaluated for their ability to purify the anti-fluorescein mAb from a mixture of commercial preparation and an E. coli cell lysate. The recovered mAb was obtained at relatively high purity (88-95%), in a wide range of target concentrations (0.66-0.02 mg/ml) within a total purification time of approximately 20 min. Substantial increase in the contamination background did not affect purity.
Assuntos
Anticorpos Monoclonais , Anticorpos/isolamento & purificação , Parvalbuminas/imunologia , Soroalbumina Bovina/imunologia , Albumina Sérica/imunologia , Anticorpos Monoclonais/isolamento & purificação , Formação de Anticorpos , Especificidade de Anticorpos , Cromatografia de Afinidade , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Parvalbuminas/isolamento & purificação , Albumina Sérica/isolamento & purificação , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
A strategy for clustering of native lipid membranes is presented. It relies on the formation of complexes between hydrophobic chelators embedded within the lipid bilayer and metal cations in the aqueous phase, capable of binding two (or more) chelators simultaneously Fig. 1. We used this approach with purple membranes containing the light driven proton pump protein bacteriorhodopsin (bR) and showed that patches of purple membranes cluster into mm sized aggregates and that these are stable for months when incubated at 19°C in the dark. The strategy may be general since four different hydrophobic chelators (1,10-phenanthroline, bathophenanthroline, Phen-C10, and 8-hydroxyquinoline) and various divalent cations (Ni(2+), Zn(2+), Cd(2+), Mn(2+), and Cu(2+)) induced formation of membrane clusters. Moreover, the absolute requirement for a hydrophobic chelator and the appropriate metal cations was demonstrated with light and atomic force microscopy (AFM); the presence of the metal does not appear to affect the functional state of the protein. The potential utility of the approach as an alternative to assembled lipid bilayers is suggested.