Large-scale Purification Research Articles

Large-scale isolation and purification of yolk immunoglobulin with different purity levels via a combination technique based on high-speed-shear crossflow membrane separation

Yolk immunoglobulin (IgY) is an excellent complement or substitute for mammalian immune globulin G, whose development is severely limited for its difficult isolation and purification. This work provides a combination technique for the large-scale isolation of IgY products with different purity levels to meet the market demand. Four levels (IgY-I to IgY-IV) of IgY products were sequentially separated and purified using water dilution, high-speed-shear crossflow membrane separation, and two-step salting out methods, during which the key limiting factors were optimized through the single factor tests. The IgY-I, with purity and yield of 30.69% and 25.06 mg/mL yolk, met the general demand for immune-fortified food for pets. IgY-II and IgY-III achieved purity of 35.84% and 66.09%, yielding 21.76 and 13.92 mg/mL yolk, respectively. Notably, the purity (96.9%) and yield (6.06 mg/mL yolk) of IgY-IV approximated or even exceeded the level of IgY isolated by IgY purification kits, and its purity was higher than that of the commercially available IgY standards. During the isolation, traditional centrifugation and filtration were replaced by continuous flow centrifugation and high-speed-shear crossflow membrane separation for continuous sample loading and sample concentration (a concentration factor of 85.37%). This is of great significance to shortening processing time, saving costs, and meeting needs of large-scale production. This technique has the advantages of high efficiency, low energy consumption, low cost, environmentally friendly, and friendly to human health by supplying IgY products with different purity levels to meet the market demands and promote the development of IgY-related industries.

Using a Bioactive Eremophila-Derived Serrulatane Scaffold to Generate a Unique Carbamate Library for Anti-infective Evaluations.

The known Eremophila microtheca-derived diterpenoid 3,7,8-trihydroxyserrulat-14-en-19-oic acid (1) was targeted for large-scale purification, as this bioactive plant compound has proven to be an attractive scaffold for semisynthetic studies and subsequent library generation. Compound 1 was converted to a selectively protected trimethyl derivative, 3-hydroxy-7,8-dimethoxyserrulat-14-en-19-oic acid methyl ester (2), using simple and rapid methylation conditions. The resulting scaffold 2 was reacted with a diverse series of commercially available isocyanates to generate an 11-membered carbamate-based library. The chemical structures of the 11 new semisynthetic analogues were fully characterized by spectroscopic and spectrometric analysis. All natural products and semisynthetic compounds were evaluated for their anthelmintic, antimalarial, and anti-HIV activities. Compound 3 was shown to elicit the greatest antiplasmodial activity of all compounds tested, with IC50 values of 4.6 and 11.6 μM against Plasmodium falciparum 3D7 and Dd2, respectively. Compound 11 showed the greatest inhibition of development to fourth-stage Haemonchus contortus larvae (L4) and induction of a skinny (Ski) phenotype (67.5% of nematodes) at 50 μM. Compound 7, which inhibited 59.0% of HIV production at 100 μg/mL, was the carbamate analogue that displayed the best antiviral activity.

Rapid Synthesis of Oxygen-Deficient MoO3–x-rGO Composites for Synergistic Photothermal Seawater Desalination and Photocatalytic Sterilization

The development of efficient photothermal desalination devices is of great significance in solving the problem of freshwater shortage. The design and preparation of efficient photothermal materials with a broad light absorption range have been extensively studied. However, the performance of photothermal catalysts for synergistic purification of complex seawater is still facing significant challenges. Therefore, we report the rapid preparation of MoO3–x-rGO composites with synergistic photothermal and photocatalytic performance by the solid-phase microwave thermal shock method. The superhot spots created on the surface of microwave-reduced rGO induce the formation of MoO3–x with oxygen vacancies. Due to the localized surface plasmon resonance (LSPR) effect, the near-infrared photothermal and photocatalytic properties of MoO3–x-rGO catalysts are greatly improved. When the optimal composite catalyst (GMW-3) is illuminated by one sunlight, the seawater evaporation rate and photothermal conversion efficiency can reach 1.58 kg m–2 h–1 and 87.2%, respectively. Meanwhile, the GMW-3 composite evaporator shows excellent antibacterial performance under the synergistic effect of photothermal and photocatalysis during the evaporation process. These findings expand a new insight and platform to design efficient catalysts for large-scale seawater desalination and purification.

Non-Affinity Purification of Antibodies.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry.Graphical

Structure study reveals active and inactive conformations of protein kinase C B1.

A human cell-based recombinant protein expression system is advantageous for large scale protein expression and purification as it provides native and nearly ideal conditions for correct folding and post-translational modification of protein products. Recently, we developed an affinity tag system using llama single-domain antibodies (a.k.a. nanobodies) generated against the fluorescent proteins YFP and mCherry. These nanobodies are highly efficient, with high binding capacity while exhibiting binding affinities that are down to the sub-nanomolar Kd, serving as ideal affinity-matrices for routine protein expression and purification in a mammalian system. These mCherry-tag and YFP-tag systems have enabled us to successfully express and purify numerous recombinant human proteins with productive yields and high quality. Here, we utilize the YFP-tag system to express and purify recombinant full-length human Protein Kinase C βI (PKCβI) using HEK293F cells. We have determined structures of purified PKCβI protein using X-ray crystallography that define two distinct conformations of the enzyme, providing structural insights behind their inhibition and activation mechanisms.

Identification and characterization of E266Q mutation in Nsp15 endoribonuclease from SARS-CoV-2 Epsilon variant.

SARS-CoV-2 is responsible for the ongoing global pandemic of COVID-19. Nsp15, an endoribonuclease from SARS-CoV-2 plays active roles in immune evasion and hence emerged as a drug target for COVID-19. Here we report the identification and characterization of a high frequency mutation in Nsp15 from the SARS-CoV-2 variant, Epsilon. First detected in California, USA in July 2020, Epsilon exhibited increased transmissibility compared to other SARS-CoV-2 variants circulating at the time. We performed multiple sequence alignment of 126 genomes of epsilon and identified four non-synonymous amino acid changes in Nsp15 (V66L, A81V, D183N and E266Q). We created these four single mutants to study their impact on Nsp15 catalysis. We also created a catalytically inactive mutant (H234A) for comparative analysis. Initial protein expressions revealed, E266Q which had a mutation rate of 4.76% exhibited very low expression levels compared to wild type (WT) whereas the inactive H234A exhibited very higher expression levels. This observation was reiterated by large-scale protein purification as well. We hypothesize that being an endoribonuclease, Nsp15 might be cleaving its own mRNA during recombinant expression, thus the activity levels of E266Q, WT, and H234A directly correlate to their corresponding expression levels. We performed a preliminary activity assay which demonstrated catalytic efficiency of E266Q is equal to or even slightly higher than WT. Enzyme kinetics experiments are underway to confirm this phenomenon. We also designed a new mutation E266A to further explore the effect of the E266 residue in Nsp15 activity. Currently, we are pursuing comparative structural and functional studies on E266Q, E266A, WT, and H234A to understand how E266Q mutation renders increased catalytic activity to Nsp15 and influences Epsilon's disease transmissibility.

Scalable, Highly Pure, and Diameter-Sorted Boron Nitride Nanotube by Aqueous Polymer Two-Phase Extraction.

Boron nitride nanotube (BNNT) has attracted recent attention owing to its exceptional material properties; yet, practical implementation in real-life applications has been elusive, mainly due to the purity issues associated with its large-scale synthesis. Although different purification methods have been discussed so far, there lacks a scalable solution method in the community. In this work, a simple, high-throughput, and scalable purification of BNNT is reported via modification of an established sorting technique, aqueous polymer two-phase extraction. A complete partition mapping of the boron nitridespecies is established, which enables the segregation of the highly pure BNNT with a major impurity removal efficiency of > 98%. A successful scaling up of the process is illustrated and provides solid evidence of its diameter sorting behavior. Last, towards its macroscopic assemblies, a liquid crystal of the purified BNNT is demonstrated. The effort toward large-scale solution purification of BNNT is believed to contribute significantly to the macroscopic realization of its exceptional properties in the near future.

Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies.

Toxin-antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I-VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods. Key features Detailed protocol for the large-scale purification of ToxIN type III toxin-antitoxin complexes from E. coli. The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA. Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones. The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.

Extracellular Vesicle Isolation by a Tangential-Flow Filtration-Based Large-Scale Purification Method.

Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium. Inserting the TFF step before PEG precipitation removes proteins, which may aggregate in subsequent steps and co-purify with EVs.

Genetic engineering approaches to study of the opines of natural GMOs

A few dozen naturally transgenic plants have been described to date [1]. Many of them contain DNA-sequences homologous to opine biosynthesis genes. Chemically, opines fall into two major structural classes: secondary amine derivatives and sugar-phosphodiesters [2]. The concentration of these molecules in the crude plant extract of nGMOs could be less than 1 pmol, which makes it difficult to study them. However, structure clarification of plant opines reveal their function in plants. The aim of our work is the development of protocol for large scale production, purification and definition of the chemical structure of plant opines.
 The approach includes amplification of full-length opine synthase gene by PCR and cloning it using pENTR/D-TOPO Cloning Kit (Thermo Fisher) according to its manual; subcloning in pDest527 using LR Clonase II Plus enzyme (Thermo Fisher) according to its manual; transformation NiCo21(DE3) chemically competent E. coli cells with obtained recombinant plasmids. After the confirmation of transgenic nature, the E. coli cells should be grown on medium with ampicillin 100 mg/l to an optical density at 600 nm 0.50.6. Then an equal volume of LB medium with 1 mM isopropylthio--galactoside (IPTG) should be added for induction opine synthesis. In case of synthesis of sugar-phosphodiesters, cultural media should also contain 0.02 M arabinose, glucose, and sucrose, respectively. Cells are cultivated during 4 hours for induction of opine synthesis. After that cells are pelleted by centrifugation for 10 minutes at 5000 rpm/min. The original strain and cells grown on the medium without IPTG are used as controls.
 The cells are extracted with 80% methanol. The culture liquids are evaporated and redissolved in 80% methanol. The opines are separated by normal-phase chromatography using a CHROMABOND Flash BT. Thin-layer chromatography is used to analyze the fractions, and to confirm the component opines-like compounds. High-resolution mass spectra are recorded on a Bruker micrOTOF 10223 mass spectrometer (electrospray ionization), eluent 80% MeOH. The 31P NMR spectra are acquired on a Bruker Avance 400 spectrometer (400, 100, and 162 MHz, respectively). The 1H spectra are analyzed in D2O, with residual solvent signals (7.26 ppm for 1H nuclei) as the internal reference. The 31P NMR is measured relative to H3PO4.
 The one phosphoric acid residue in the opine structure was confirmed by NMR spectroscopy.
 We applied this method to characterize agrocinopine A-like compound in Nicotiana and propose it to produce opine-like plant compounds for further biochemical analysis.
 This research was funded the Ministry of Science and Higher Education of the Russian Federation in accordance with the agreement No. 075-15-2022-322.
 This work was partially carried out using equipments of the Saint Petersburg State University Chemical Analysis and Materials Research Centre.

Oncolytic Urabe mumps virus: A promising virotherapy for triple-negative breast cancer

Historically, the clinical utility of oncolytic virotherapy as a treatment for a wide range of cancer types was first demonstrated by three pilot human clinical trials conducted in Japan in the 1970s and 1980s using a wild-type Urabe mumps virus (MuV) clinical isolate. Using a sample of the actual original oncolytic Urabe MuV clinical trial virus stock (MuV-U-Japan) used in these Japanese clinical trials, we found that MuV-U-Japan consisted of a wide variety of very closely related Urabe MuVs that differed by an average of only three amino acids. Two MuV-U-Japan isolates, MuV-UA and MuV-UC, potently killed a panel of established human breast cancer cell lines invitro, significantly extended survival of nude mice with human triple-negative breast cancer (TNBC) MDA-MB-231 tumor xenografts invivo, and demonstrated significant killing activity against breast cancer patient-derived xenograft (PDX) cell lines grown as 3D organoids, including PDXs from patients resistant to anthracycline- and taxane-based chemotherapy. We also report success in developing a large-scale MuV-U production and purification process suitable for supporting Investigational New Drug applications for clinical trials. This study demonstrates the suitability of the MuV-UC virus for translation to modern clinical trials for treating patients with TNBC.

Enrichment of nervonic acid in Acer truncatum Bunge oil by combination of two-stage molecular distillation, one-stage urea complexation and five-stage solvent crystallization

Nervonic acid is the world's first and only potent substance that can repair damaged nerve fibers and promote nerve cell regeneration with high nutritional value. The wide variety of fatty acids in plant oils and fats with similar structures makes the large-scale separation and purification of high-purity nervonic acid very difficult. A new combined process of molecular distillation, urea inclusion and solvent crystallization was established to prepare high-purity nervonic acid with the mixed fatty acids obtained after saponification and acidification of Acer truncatum Bunge oil as raw materials. First, according to the difference in the mean free path of fatty acids, molecular distillation was used to separate and remove C16 saturated fatty acid of palmitic acid and four C18–C20 fatty acids of stearic, oleic, linoleic, and linolenic acids. The content of C16–C20 fatty acids decreased from 72.92% to 19.22% after two-stage molecular distillation processes, in which the contents of saturated fatty acid of palmitic acid decreased to about 0.5%. Then, according to the difference in carbon chain length and saturation of fatty acid, the contents of C22–C24 saturated fatty acids of tetracosanoic and docosanoic acids decreased to 0.21% and 0.07% by urea inclusion with urea/free fatty acid preparation by saponification (SPOMFs) ratio as 0.6. In addition, all saturated fatty acids were basically separated. Finally, according to the difference in the solubility of fatty acids in solvents, the C18–C20 unsaturated fatty acids of oleic, linoleic, and linolenic acids and C22 unsaturated fatty acid of erucic acid were removed by solvent crystallization. The content of C18–C20 unsaturated fatty acids decreased to less than 5% with pentanol as the solvent after the first stage solvent crystallization. The content of erucic acid decreased to 3.47% with anhydrous ethanol as the solvent after the second to fifth stage solvent crystallization. The combined process of molecular distillation, urea inclusion and low temperature crystallization innovatively adopted an efficient, simple and easy-to-industrial solvent crystallization method to separate erucic and nervonic acids, obtaining nervonic acid with purity of 96.53% and final yield of 47.99%.

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

Parallelized continuous flow dielectrophoretic separation ofDNA.

Numerous microfluidic separation applications have been shown in the past years providing a fast analysis of biological samples like DNA or proteins. Microfluidic separation based on dielectrophoresis (DEP), that is the migration of a polarizable object in an inhomogeneous electric field, provides numerous advantages. However, the main drawback of DEP separation devices is that they are not sufficient for large-scale sample purification due to the lack of high sample throughput. In this work, we present for the first time a microfluidic device with two parallelized dielectrophoretic separations of (biological) samples smaller than 1µm. The separation is carried out by means of insulator-based DEP, that is an insulating ridge reduced the flow through height and thus created a nanoslit at which the selective DEP forces occur. The device consists of a cross injector, two parallel operation regions and separate harvesting reservoirs where the samples are collected. Each DEP operation region contains an insulating ridge. We successfully demonstrate the separation of 100 and 40nm beads and 10 and 5kbp DNA with a separation purity of more than 80%. This states the proof-of-concept for up-scaling of dielectrophoretic separation by parallelization. As the present technique is virtually label-free, it offers a fast purification, for example in the production of gene vaccines.

Development and scale-up of oligo-dT monolithic chromatographic column for mRNA capture through understanding of base-pairing interactions

Oligo-deoxythymidilic acid (OdT) probes conjugated to solid-phase supports exhibit high affinity for poly-adenylated mRNA (target) through high-affinity base-pairing interactions. Here we report the development of a OdT-functionalized chromatographic monolith for purification of polyadenylated mRNA and development of purification methods to support large-scale purification of mRNA-based therapeutics. We report the development of a chromatographic assay based on a synthetic oligo-deoxyadenylic acid chain of 10 or 20 nucleotides (OdA10 and OdA20) as a surrogate for polyadenylated mRNA, which was used for optimization of the OdT affinity column (i.e. the amount and structure of OdT immobilized and monolithic channel size). OdA hybridization to OdT monoliths correlated well with the amount of immobilized OdT, while an in-depth analysis revealed that hybridization yield decreased with increasing size of the target, temperature and probe surface coverage. OdA hybridization kinetics was flow rate-independent, confirming convection-based mass transport within the monolith’s channels. The demonstrated steep adsorption isotherms enable chromatographic capture of even highly diluted OdA-containing molecules. Dynamic binding capacity for model mRNA was independent of OdT length and amount of immobilized OdT probes above a critical threshold but was highly influenced by the composition of the binding buffer and mRNA residence time. We demonstrated the scalability of the mRNA purification process with OdT monoliths from 0.1 mL to at least 800 mL bed volume, paving the way for manufacturing processes on OdT monoliths with 40 L bed volume.

Expression strategies for the efficient synthesis of antimicrobial peptides in plastids

Antimicrobial peptides (AMPs) kill microbes or inhibit their growth and are promising next-generation antibiotics. Harnessing their full potential as antimicrobial agents will require methods for cost-effective large-scale production and purification. Here, we explore the possibility to exploit the high protein synthesis capacity of the chloroplast to produce AMPs in plants. Generating a large series of 29 sets of transplastomic tobacco plants expressing nine different AMPs as fusion proteins, we show that high-level constitutive AMP expression results in deleterious plant phenotypes. However, by utilizing inducible expression and fusions to the cleavable carrier protein SUMO, the cytotoxic effects of AMPs and fused AMPs are alleviated and plants with wild-type-like phenotypes are obtained. Importantly, purified AMP fusion proteins display antimicrobial activity independently of proteolytic removal of the carrier. Our work provides expression strategies for the synthesis of toxic polypeptides in chloroplasts, and establishes transplastomic plants as efficient production platform for antimicrobial peptides.

A simple scheme for large scale purification of urine – Derived Bence Jones Kappa protein

We have investigated and optimized purification process, suitable for industrial scale, to obtain high purity grade Bence Jones Kappa Protein (‘BJK-Protein’), while preserving its physiological properties and functions. BJK-Protein was obtained from a biological waste product i.e. human urine of renal failure patients. Isolated ‘BJK-Protein’ was analyzed by electrophoresis, western blotting, double immunodiffusion, SEC-HPLC assay and Mass Spectrometry (MS). The relative molecular mass of ‘BJK-Protein’ is 23054.2 Da. Moreover, dimer forms of ‘BJK-Protein’ were also detected in SDS-PAGE and mass spectrum corresponding to 46054.4 Da. The results of western blotting, immunoelectrophoresis, SEC-HPLC assay, and mass spectrometry analysis indicate a high purity (>99 %) of ‘BJK-Protein’. Peptide mass fingerprint analysis of ‘BJK-Protein’ yielded peptides that partially matches the known database sequences of kappa variable region (KV139_HUMAN) of immunoglobulin. This protein was found to be stable up to 20 months at 2–8 °C temperature and also found negative for major undesirable viral markers as well as bacterial endotoxin. With this purification approach, the cost of purified ‘BJK-Protein’ is significantly reduced as compared to the current market price of Kappa light chain available in international market.

Enhanced photo-Fenton assisted photocatalytic degradation of atenolol using a novel rGO embedded double Z-scheme nano-heterojunction: Mechanism, kinetics and toxicity studies

In this work, rGO-based ternary dual Z-scheme heterojunction (rGO/CuFe2O4/CdS/Bi2S3 QDs) was formulated for the effective photo-Fenton assisted photocatalytic degradation of a β-blocker, atenolol. The catalyst was synthesised via a simple co-precipitation/hydrothermal method and was characterized using XRD, XPS, FT-IR, SEM, TEM, PL, EIS, ESR, Raman and DRS. It exhibited about 76.5% degradation of atenolol in 360 min under visible light irradiation with a rate constant of 0.004 min−1 wherein the mechanism of enhanced activity was attributed to the formation of OH, h+ and photo-Fenton reaction; a possible mechanism was drafted. The intermediates were determined using GC–MS analysis delineating a plausible degradation pathway, the mineralization of atenolol being confirmed by TOC analysis. The catalyst was reusable and highly stable up to six cycles of degradation, as affirmed via XRD and XPS analysis. Further, the toxicity of the catalyst has been studied using the root cells of Allium cepa, and the degraded product and intermediates were assessed deploying ECOSAR software. Thus, the proposed work has potential which can be implemented in future for the large-scale purification of wastewater.

Efficient and Scalable Process to Produce Novel and Highly Bioactive Purified Cytosolic Crystals from Bacillus thuringiensis.

ABSTRACTBacillus thuringiensis (Bt) is a Gram-positive soil bacterium that is widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops or are disease vectors. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e.g., Cry5B (formally Cry5Ba1), target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described inactivated bacteria with cytosolic crystal(s) (IBaCC) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here, we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents, yielding purified cytosolic crystals (PCC). Cry5B PCC contains ~95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro. Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100-liter scale to produce PCC for a pilot study to treat two foals infected with the ascarid Parascaris spp. Single-dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new and attractive pharmaceutical ingredient form of Bt Cry proteins.IMPORTANCE Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies have shown they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called purified cytosolic crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins is desirable and where these methods will find great utility.