Synthetic two-dimensional (2D) protein assemblies were engineered using tandem Z-domains derived from the bacterial Protein A. Assembly was induced by introducing hexa-histidine tags to both the N- and C-termini of the tandem Z-domain ((His)6-(Z)2-(His)6) and adding equimolar Zn2+ at pH 7. Two lines of evidence suggest preservation of the Z-domain's native structure upon metal-mediated assembly: (i) far-UV circular dichroism spectroscopy; and (ii) selective binding to IgG antibodies, with no detectable interaction with IgA or IgM, consistent with the known specificity of the Z-domain. Scanning transmission electron microscopy demonstrated the formation of 2D protein assemblies exclusively in the presence of Zn2+ ions. The widespread use of His-tag engineering and the mild conditions required to assemble (His)6-(Z)2-(His)6 monomers into two-dimensional structures suggest that this approach offers a straightforward and accessible platform for the fabrication of synthetic 2D protein assemblies with potential applications in biotechnology and medicine.

Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG) that does not require expensive ligands, chromatography columns, polymers, nor membranes. Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5-15 mg/mL) in the supernatant. [(batho)3:Zn2+] complexes were the most promising, resulting in hIgG with purity ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves native secondary structure (by far UV circular dichroism spectroscopy, CD). Process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume that required only proportional increase in reagents. While Protein A chromatographic columns, the industry gold standard, have limited binding capacity, are costly and require familiarity with column maintenance, we are attempting, by our efforts, to help produce a more efficient, simple and economical purification platform.

Doubly His6-tagged mCherry red fluorescent proteins are observed to form fibers and sheets at neutral pH in the presence of no more than equimolar amounts of Zn2+ or Ni2+. These architectures, on the order of 10 μm in extent, are detected with scanning transmission electron microscopy imaging. Far ultraviolet circular dichroism spectroscopy attests to the preservation of the native secondary structure of mCherry, while the emission spectrum reveals the maintenance of the chemical environment of the fluorophore site. Two-dimensional, fluorescence microscopy images provide evidence for our assertion that the mechanism underlying protein assembly relies on [metal:chelator] conjugation, i.e., between a His6-tag and divalent cations: (a) Conjugation is reversible when competing water-soluble chelators (e.g., 5 mM EDTA, histidine or imidazole) are present; (b) Conjugation depends on pH. Below pH 6, when more than 50 % of the imidazole rings in the His6-tag are protonated, protein conjugation is suppressed. The straightforward chemistry with which our approach can be implemented, combined with its potential generality and non-denaturing properties, suggests that these fluorescent biopolymers may be suitable for enhancing the sensitivity of immunoassays and histology staining studies.

In this study, we assess an alternative Fc-fusion protein purification method that does not rely on chromatographic media or ligands. Recombinant human acetylcholinesterase, fused to the Fc domain of human IgG1 (henceforth, AChE-Fc), was purified with precipitated aromatic complexes composed of the bathophenanthroline (henceforth, batho) chelator with either Zn2+ or Cu2+ ions (i.e., [(batho)3:Zn2+] or [(batho)2:Cu2+]) in the presence of polyethylene glycol 6000 (PEG-6000). In a three-step purification process conducted at pH 7, AChE-Fc was captured by the aromatic complexes (Step 1); unbound or weakly bound protein impurities were removed with 20 mM NaCl (Step 2); and AChE-Fc was then extracted at pH 7 (Step 3) using 100 mM Na citrate buffer in 250 mM NaCl. Purified AChE-Fc was not aggregated (as determined by dynamic light scattering (DLS) and Native PAGE). However, full enzymatic activity was only preserved with the [(batho)3:Zn2+] complex. Interaction between AChE-Fc and [(batho)3:Zn2+] led to ~83-88% overall protein yield. Thirty-fold process upscaling by volume required only proportional increase in the amounts of [(batho)3:Zn2+] and PEG-6000. Efficient (95-97%) chelator recycling was achieved by recrystallization. Chelator leaching into purified AchE-Fc was estimated to be ~0.3% relative to the total amount used. Taken together, this novel procedure has the potential to provide an economical and practical avenue for the industrial purification of Fc-fusion proteins.

Background/Objectives: Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic-grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG), which does not require any expensive ligands, chromatography columns, polymers, or membranes. Methods/Results: Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5–15 mg/mL) in the supernatant. [(Batho)3:Zn2+] complexes were the most promising, resulting in hIgG with a purity of ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves the native secondary structure (by far UV circular dichroism spectroscopy, CD). The process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume, which required only proportional increases in reagents. Conclusions: Although Protein A chromatographic columns, the industry gold standard, have a limited binding capacity, are costly, and require familiarity with column maintenance, we are attempting, by our efforts, to help to produce a more efficient, simple, and economical purification platform.

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.

The term supramolecular polymer has been applied to polymeric materials in which the individual units, i.e., building blocks-are bound to each other via noncovalent interactions, including electrostatic or hydrogen bonding, as well as metal-ligand conjugation. The building blocks are generally low molecular weight amphiphiles. Methods for preparing biopolymers based on non-toxic, metal-ligand conjugation have been little studied; however, they offer significant potential for tuning the response of biologically relevant macromolecules. In this communication, we characterize the assembly and morphology of supramolecular biopolymers in which the building blocks are low- or medium-molecular weight globular proteins-ubiquitin and Cas9-interacting via metal-ligand conjugation. In each case, the protein gene was expressed in cell culture with the addition of hexa-His/linkers at both the N and C termini. Divalent cations investigated were Zn2+ and Ni2+. We observe in cryo-TEM imaging an absolute requirement for divalent cations for the formation of supramolecular biopolymers. In the presence of Ni2+, 1D assembled fibers are predominant, while with Zn2+, the more frequently detected structures are sheet-like. We use gel electrophoresis and CD spectroscopy to monitor possible secondary and tertiary structural changes in the protein building blocks during conjugation.

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 (∼90%, 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.

As downstream purification and separation technologies progress towards raising the concentration of therapeutic-grade monoclonal antibodies (mAbs) in cell culture, downstream processing has begun to face increased difficulty in efficiently coping with such high immunoglobulin G (IgG) titers (<= 25 mg mL(-1)). In the current study, we demonstrate the ability of a non-chromatographic, ligand-free procedure to recover almost quantitatively (84-99% yield, by densitometry) polyclonal, human IgG present at high concentrations (15-25 mg mL(-1)) in E. coli lysate. Instead of chromatographic media and columns, we use conjugated, mixed-micelles comprising non-ionic detergents, tyrosine monomers, and the amphiphilic [(bathophenanthroline)(3):Fe2+] complex. Capture and extraction processes are performed at pH 6.5-7, thereby avoiding antibody exposure to acidic, potentially denaturing conditions. Recovered IgG is monomeric as determined by dynamic light scattering (DLS). Process upscaling from 0.1 to 5 mL requires only proportional increase in all reagents and does not affect overall yield or antibody purity.

Specific conjugation of decyl β-D-maltoside (DM) or dodecyl β-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 (His 1 -C10) and Ni 2+ 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: Zn 2+ , Fe 2+ , Cu 2+ lead to dark precipitates rather than colorless globules; while Mg 2+ , Ca 2+ 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 His 1 -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 His 1 -tagged DM or DDM micelles for promoting crystallization of integral membrane proteins is discussed.

Immunoglobulin-G (IgG) (∼150 kDa) antibodies confer longer term immunity against bacterial or viral infections than the heavier IgM's (∼900 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.

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.

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.

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.

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.

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 similar to 80 % were also observed when Ab concentrations were increased from 1 mg/mL to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies, are discussed.

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.

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.

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 immtmoglobulins (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 (pH = 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.

We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose-synthesized, peptide amphiphiles, C-10-(Asp)(5) and C-10-(Lys)(5). Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C-10-(Asp)(5) are conjugated (a) via a water-soluble, penta-Lys mediator or (b) to micelles containing the C-10-(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.