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Chemical Reviews Mar 2024Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological... (Review)
Review
Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological activity. In this field, ionic liquids (ILs) have gained momentum in the past years, with a relevant number of works reporting their successful use to dissolve, stabilize, extract, and purify proteins. Different approaches in protein-IL systems have been reported, namely, proteins dissolved in () neat ILs, () ILs as co-solvents, () ILs as adjuvants, () ILs as surfactants, () ILs as phase-forming components of aqueous biphasic systems, and () IL-polymer-protein/peptide conjugates. Herein, we critically analyze the works published to date and provide a comprehensive understanding of the IL-protein interactions affecting the stability, conformational alteration, unfolding, misfolding, and refolding of proteins while providing directions for future studies in view of imminent applications. Overall, it has been found that the stability or purification of proteins by ILs is bispecific and depends on the structure of both the IL and the protein. The most promising IL-protein systems are identified, which is valuable when foreseeing market applications of ILs, e.g., in "protein packaging" and "detergent applications". Future directions and other possibilities of IL-protein systems in light-harvesting and biotechnology/biomedical applications are discussed.
Topics: Ionic Liquids; Proteins; Solvents; Water; Polymers
PubMed: 38437627
DOI: 10.1021/acs.chemrev.3c00551 -
Protein Expression and Purification Jun 2024Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein...
Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.
Topics: Trypanosoma cruzi; Cysteine Endopeptidases; Epitopes; Escherichia coli; Membrane Proteins
PubMed: 38423156
DOI: 10.1016/j.pep.2024.106458 -
ACS Bio & Med Chem Au Feb 2024Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables...
Deciphering the Synthetic and Refolding Strategy of a Cysteine-Rich Domain in the Tumor Necrosis Factor Receptor (TNF-R) for Racemic Crystallography Analysis and d-Peptide Ligand Discovery.
Many cell-surface receptors are promising targets for chemical synthesis because of their critical roles in disease development. This synthetic approach enables investigations by racemic protein crystallography and ligand discovery by mirror-image methodologies. However, due to their complex nature, the chemical synthesis of a receptor can be a significant challenge. Here, we describe the chemical synthesis and folding of a central, cysteine-rich domain of the cell-surface receptor tumor necrosis factor 1 which is integral to binding of the cytokine TNF-α, namely, TNFR-1 CRD2. Racemic protein crystallography at 1.4 Å confirmed that the native binding conformation was preserved, and TNFR-1 CRD2 maintained its capacity to bind to TNF-α ( ≈ 7 nM). Encouraged by this discovery, we carried out mirror-image phage display using the enantiomeric receptor mimic and identified a d-peptide ligand for TNFR-1 CRD2 ( = 1 μM). This work demonstrated that cysteine-rich domains, including the central domains, can be chemically synthesized and used as mimics for investigations.
PubMed: 38404743
DOI: 10.1021/acsbiomedchemau.3c00060 -
Molecular Biology Reports Feb 2024SLC38A2 is a ubiquitously expressed Na-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II...
BACKGROUND
SLC38A2 is a ubiquitously expressed Na-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy.
METHODS AND RESULTS
The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane.
CONCLUSIONS
The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.
Topics: Humans; Amino Acid Transport System A; Membrane Transport Proteins
PubMed: 38393484
DOI: 10.1007/s11033-023-08976-3 -
Advanced Materials (Deerfield Beach,... May 2024Cytokines are powerful in cancer immunotherapy, however, their therapeutic potential is limited by the severe systemic toxicity. Here a potent strategy to reduce the...
Cytokines are powerful in cancer immunotherapy, however, their therapeutic potential is limited by the severe systemic toxicity. Here a potent strategy to reduce the toxicity of systemic cytokine therapy by delivering its denatured form using a finely designed nanochaperone, is described. It is demonstrated that even if the denatured protein cargos are occasionally released under normal physiological conditions they are still misfolded, while can effectively refold into native states and release to function in tumor microenvironment. Consequently, the systemic toxicity of cytokines is nearly completely overcome. Moreover, an immunogenic cell death (ICD)-inducing chemotherapeutic is further loaded and delivered to tumor using this nanochaperone to trigger the release of tumor-associated antigens (TAAs) that are subsequently captured in situ by nanochaperone and then reflows into lymph nodes (LNs) to promote antigen cross-presentation. This optimized personalized nanochaperone-vaccine demonstrates unprecedented suppressive effects against large, advanced tumors, and in combination with immune checkpoint blockade (ICB) therapy results in a significant abscopal effect and inhibition of postoperative tumor recurrence and metastasis. Hence, this approach provides a simple and universal delivery strategy to reduce the systemic toxicities of cytokines, as well as provides a robust personalized cancer vaccination platform, which may find wide applications in cancer immunotherapy.
Topics: Animals; Humans; Mice; Antigens, Neoplasm; Cancer Vaccines; Cell Line, Tumor; Immunogenic Cell Death; Immunotherapy; Interleukin-12; Nanoparticles; Nanostructures; Neoplasms; Protein Folding; Tumor Microenvironment
PubMed: 38387609
DOI: 10.1002/adma.202309927 -
Organic & Biomolecular Chemistry Mar 2024Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core...
Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.
Topics: Humans; Capsid; Capsid Proteins; Hepatitis B virus; Hepatitis B; Viral Core Proteins; Nucleic Acids; Virus Replication; Antiviral Agents
PubMed: 38358380
DOI: 10.1039/d3ob02099a -
Molecular Biotechnology Feb 2024Vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding...
Vascular endothelial growth factor A (VEGF-A) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A receptor-binding domain (VEGFA-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA-RBD. 25 ng sKDR1-3 and 20 ng VEGFA-RBD showed strong binding. sKDR1-3 bound to VEGFA-RBD and VEGF-A with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.
PubMed: 38349458
DOI: 10.1007/s12033-024-01057-1 -
Nanoscale Mar 2024We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of β-cyclodextrin (β-CD) covalently embedded in an ultrathin...
We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of β-cyclodextrin (β-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the β-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.
Topics: Cyclodextrins; Protein Folding; Molecular Chaperones; Surface-Active Agents
PubMed: 38349359
DOI: 10.1039/d3nr06044f -
Microbial Cell Factories Feb 2024The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive...
BACKGROUND
The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult.
RESULTS
We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography.
CONCLUSIONS
Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications.
Topics: Escherichia coli; Recombinant Proteins; Inclusion Bodies; Disulfides
PubMed: 38347541
DOI: 10.1186/s12934-024-02316-1 -
Journal of Molecular Biology Mar 2024Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use...
Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.
Topics: Escherichia coli; Exoribonucleases; Kinetics; Protein Folding; Protein Multimerization; Silent Mutation; Cell Membrane
PubMed: 38341172
DOI: 10.1016/j.jmb.2024.168487