An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model.

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD's eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell's receptor, with a binding constant of 7.9 × 10-9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.

Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources.

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.

Expression and characterization of recombinant rattusin, an α-defensin-related peptide with a homodimeric scaffold formed by intermolecular disulfide exchanges.

Rattusin is an α-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations. Therefore, to develop new antibiotics based on rattusin, structural and functional studies of rattusin should be performed. For this purpose, large amounts of linear rattusin precursor must be obtained through appropriate preparation methods. Therefore, we established a mass production technique for linear rattusin by using recombinant protein expression and purification procedures. We verified that structure and activity of the recombinant rattusin are identical to the chemically synthesized rattusin. The described method for producing recombinant rattusin provides a high yield of rattusin, which can be used to study the biochemical and functional properties of rattusin and for the development of rattusin-based peptide antibiotics.

Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The ß-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling.

Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.

Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.

A quantitative assay for binding and inhibition of Plasmodium falciparum Erythrocyte Binding Antigen 175 reveals high affinity binding depends on both DBL domains.

Plasmodium falciparum Erythrocyte Binding Antigen 175 (PfEBA-175) engages Glycophorin A (GpA) on erythrocytes during malaria infection. The two Duffy binding like domains (F1 and F2) of PfEBA-175 that form region II (RII) are necessary for binding GpA, and are the target of neutralizing antibodies. Recombinant production of RII in Pichia pastoris and baculovirus has required mutations to prevent aberrant glycosylation or deglycosylation resulting in modifications to the protein surface that may affect antibody recognition and binding. In this study, we developed a recombinant system in Escherichia coli to obtain RII and F2 without mutations or glycosylation through oxidative refolding. The system produced refolded protein with high yields and purity, and without the need for mutations or deglycosylation. Biophysical characterization indicated both proteins are well behaved and correctly folded. We also demonstrate the recombinant proteins are functional, and develop a quantitative functional flow cytometry binding assay for erythrocyte binding ideally suited to measure inhibition by antibodies and inhibitors. This assay showed far greater binding of RII to erythrocytes over F2 and that binding of RII is inhibited by a neutralizing antibody and sialyllactose, while galactose had no effect on binding. These studies form the framework to measure inhibition by antibodies and small molecules that target PfEBA-175 in a rapid and quantitative manner using RII that is unmodified or mutated. This approach has significant advantages over current methods for examining receptor-ligand interactions and is applicable to other erythrocyte binding proteins used by the parasite.

Oxidative refolding by Copper-catalyzed air oxidation consistently increases the homogeneity and activity of a Novel Interleukin-2 mutein.

Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.

Production of Metridia Luciferase in Native Form by Oxidative Refolding from E. coli Inclusion Bodies.

The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies. The method includes (i) purification of MLuc inclusion bodies, solubilization of the aggregated form with full reduction of disulfide bonds, and refolding to the native state using a glutathione redox system in the presence of arginine and Cu2+ ions and (ii) chromatographic purification of MLuc and its functional assessment in terms of activity. We introduce the empirical, optimal conditions for oxidative refolding and subsequent purification of MLuc, with its basic properties taken into account. We believe that this protocol is adaptable for a large-scale harvest of other natively folded copepod luciferases as well as other disulfide-rich recombinant proteins from E. coli inclusion bodies.

Redox responsive albumin autogenic nanoparticles for the delivery of cancer drugs.

The present study explores preparation and characterization of redox sensitive albumin autogenic nanoparticles (ANPs) for drug delivery applications. Human serum albumin nanoparticles are prepared by desolvation method. The particles are stabilized through self-crosslinking and no external stabilizers are involved in the preparation. ANPs are then subjected to Camptothecin (CPT) drug loading. Experiments on in vitro and in vivo release profile, cytotoxic and cytocompatability, hemocompatability, blood clearance, tracking and bio imaging are studied in detail. The redox sensitive and drug release properties of ANPs studied in the presence of glutathione. Results on the physical, chemical and instrumental characterization warrant the property of the nanoparticles. ANPs obtained in the present study is biocompatible, biodegradable, effectively entangle the chosen drug, release the drug in the controlled manner, sensitive to reducing environment, nil toxicity and appreciable uptake by cells. In the current scenario on the requirement of a drug carrier with redox sensitive property to encounter cancer cells, the results of the present study on albumin nanoparticles with redox sensitivity is smart and pave the way in the cancer therapeutics.

The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λmax=480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies.