Optimized rapid production of recombinant secreted proteins in CHO cells grown in suspension: The case of RBD.

Chinese Hamster Ovary cells (CHO) have become the most common workhorse for the commercial production of therapeutic proteins, as well as for the production of recombinant proteins for biomedical research. The ability to grow at high density in suspension, the adaptability to serum free media, and the ease transfection and scale up, made CHO cell line highly productive and robust for large-scale production. Here, we present an optimized workflow used to successfully express and purify a number of human proteins with a yield up to 5 mg/L of culture. The entire protocol, from the synthetic gene design to the assessment of purified protein quality, can be completed in 2 weeks. The established cell culture platform has been efficiently adapted to rapidly produce the receptor-binding domain (RBD) in SARS-CoV-2 S protein, a protein required by many laboratories in 2020 to better understand the initial step of infection related to COVID-19 pandemic. An overall yield of 2 mg of high quality soluble RBD per liter of culture was obtained, a production 10-times cheaper than commercial preparations, this representing an intriguing strategy for future challenges.

Silk fibroin microgels as a platform for cell microencapsulation.

Cell microencapsulation has been utilized for years as a means of cell shielding from the external environment while facilitating the transport of gases, general metabolites, and secretory bioactive molecules at once. In this light, hydrogels may support the structural integrity and functionality of encapsulated biologics whereas ensuring cell viability and function and releasing potential therapeutic factors once in situ. In this work, we describe a straightforward strategy to fabricate silk fibroin (SF) microgels (µgels) and encapsulate cells into them. SF µgels (size ≈ 200 µm) were obtained through ultrasonication-induced gelation of SF in a water-oil emulsion phase. A thorough physicochemical (SEM analysis, and FT-IR) and mechanical (microindentation tests) characterization of SF µgels were carried out to assess their nanostructure, porosity, and stiffness. SF µgels were used to encapsulate and culture L929 and primary myoblasts. Interestingly, SF µgels showed a selective release of relatively small proteins (e.g., VEGF, molecular weight, MW = 40 kDa) by the encapsulated primary myoblasts, while bigger (macro)molecules (MW = 160 kDa) were hampered to diffusing through the µgels. This article provided the groundwork to expand the use of SF hydrogels into a versatile platform for encapsulating relevant cells able to release paracrine factors potentially regulating tissue and/or organ functions, thus promoting their regeneration.

High-Throughput Strategy for Glycine Oxidase Biosensor Development Reveals Glycine Release from Cultured Cells.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 µM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 µM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 µM) using the GO H244K biosensor.

An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

Expression and purification of the human tumor suppressor protein RNASET2 in CHO-S cells.

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an "alarmin" signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO-S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO-S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth.

D-amino acids in foods.

With the only exception of glycine, all amino acids exist in two specular structures which are mirror images of each other, called D-(dextro) and L-(levo) enantiomers. During evolution, L-amino acids were preferred for protein synthesis and main metabolism; however, the D-amino acids (D-AAs) acquired different and specific functions in different organisms (from playing a structural role in the peptidoglycan of the bacterial cell wall to modulating neurotransmission in mammalian brain). With the advent of sophisticated and sensitive analytical techniques, it was established during the past few decades that many foods contain considerable amounts of D-AAs: we consume more than 100 mg of D-AAs every day. D-AAs are present in a variety of foodstuffs, where they fulfill a relevant role in producing differences in taste and flavor and in their antimicrobial and antiaging properties from the corresponding L-enantiomers. In this review, we report on the derivation of D-AAs in foods, mainly originating from the starting materials, fermentation processes, racemization during food processing, or contamination. We then focus on leading-edge methods to identify and quantify D-AAs in foods. Finally, current knowledge concerning the effect of D-AAs on the nutritional state and human health is summarized, highlighting some positive and negative effects. Notwithstanding recent progress in D-AA research, the relationships between presence and nutritional value of D-AAs in foods represent a main scientific issue with interesting economic impact in the near future.

PEG-DAAO conjugate: A promising tool for cancer therapy optimized by protein engineering.

The flavoenzyme D-amino acid oxidase (DAAO) represents a potentially good option for cancer enzyme prodrug therapy as it produces H2O2 using D-amino acids as substrates, compounds present at low concentration in vivo and that can be safely administered to regulate H2O2 production. We optimized the cytotoxicity of the treatment by: i) using an efficient enzyme variant active at low O2 and D-alanine concentrations (mDAAO); ii) improving the stability and half-life of mDAAO and the enhanced permeability and retention effect by PEGylation; and iii) inhibiting the antioxidant cellular system by a heme oxygenase-1 inhibitor (ZnPP). A very low amount of PEG-mDAAO (10 mU, 50 ng of enzyme) induces cytotoxicity on various tumor cell lines. Notably, PEG-mDAAO seems well suited for in vivo evaluation as it shows the same cytotoxicity at air saturation (21%) and 2.5% O2, a condition resembling the microenvironment found in the central part of tumors.

Advances in Enzymatic Synthesis of D-Amino Acids.

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these "unnatural" molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

Biosensors for D-Amino Acids: Detection Methods and Applications.

D-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace.

Intrinsic antimicrobial properties of silk spun by genetically modified silkworm strains.

The domesticated silkworm, Bombyx mori, is a fundamental insect for silk industry. Silk is obtained from cocoons, protective envelopes produced during pupation and composed of single raw silk filaments secreted by the insect silk glands. Currently, silk is used as a textile fibre and to produce new materials for technical and biomedical applications. To enhance the use of both fabrics and silk-based materials, great efforts to obtain silk with antimicrobial properties have been made. In particular, a convincing approach is represented by the enrichment of the textile fibre with antimicrobial peptides, the main effectors of the innate immunity. To this aim, silkworm-based transgenic techniques appear to be cost-effective strategies to obtain cocoons in which antimicrobial peptides are integrated among the silk proteins. Recently, cocoons transgenic for a recombinant silk protein conjugated to the silkworm Cecropin B antimicrobial peptide were obtained and showed enhanced antibacterial properties (Li et al. in Mol Biol Rep 42:19-25, https://doi.org/10.1007/s11033-014-3735-z , 2015a). In this work we used the piggyBac-mediated germline transformation to generate several transgenic B. mori lines able to overexpress Cecropin B or Moricin antimicrobial peptides at the level of the silk gland. The derived cocoons were characterised by increased antimicrobial properties and the resulting silk fibre was able to inhibit the bacterial growth of the Gram-negative Escherichia coli. Our results suggest that the generation of silkworm overexpressing unconjugated antimicrobial peptides in the silk gland might represent an additional strategy to obtain antimicrobial peptide-enriched silk, for the production of new silk-based materials.

Binding Residence Time through Scaled Molecular Dynamics: A Prospective Application to hDAAO Inhibitors.

Traditionally, a drug potency is expressed in terms of thermodynamic quantities, mostly Kd, and empirical IC50 values. Although binding affinity as an estimate of drug activity remains relevant, it is increasingly clear that it is also important to include (un)binding kinetic parameters in the characterization of potential drug-like molecules. Herein, we used standard in silico screening to identify a series of structurally related inhibitors of hDAAO, a flavoprotein involved in schizophrenia and neuropathic pain. We applied a novel methodology, based on scaled molecular dynamics, to rank them according to their residence times. Notably, we challenged the application in a prospective fashion for the first time. The good agreement between experimental residence times and the predicted residence times highlighted the procedure's reliability in both predictive and refinement scenarios. Additionally, through further inspection of the performed simulations, we substantiated a previous hypothesis on the involvement of a protein loop during ligand unbinding.

Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity.

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn2+ ions: kinetic parameters for H2O2, Mn2+, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63-65 °C), is stable at pH 6-7, and maintains a large part of the starting activity following incubation for 24 h at 25-37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn2+, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-ß-guaiacyl ether (GGE), i.e., the Cα-Cß bond of the dimeric lignin model molecule of ß-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 µmol/min mg enzyme.

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens.

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.

Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea.

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.

Unveiling the Atomic-Level Determinants of Acylase-Ligand Complexes: An Experimental and Computational Study.

The industrial production of higher-generation semisynthetic cephalosporins starts from 7-aminocephalosporanic acid (7-ACA), which is obtained by deacylation of the naturally occurring antibiotic cephalosporin C (CephC). The enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase has attracted industrial interest because of the prospects of simplifying the process and reducing costs. We recently enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach and solved the crystal structures of wild-type VAC and the H57ßS-H70ßS VAC double variant. In the present work, experimental measurements on several CephC derivatives and six VAC variants were carried out, and the binding of ligands into the VAC active site was investigated at an atomistic level by means of molecular docking and molecular dynamics simulations and analyzed on the basis of the molecular geometry of encounter complex formation and protein-ligand potential of mean force profiles. The observed significant correlation between the experimental data and estimated binding energies highlights the predictive power of our computational method to identify the ligand binding mode. The present experimental-computational study is well-suited both to provide deep insight into the reaction mechanism of cephalosporin C acylase and to improve the efficiency of the corresponding industrial process.

Evolution of histamine oxidase activity for biotechnological applications.

Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7 mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity.

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57ßS/H70ßS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a ß-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser(1ß), is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

Revolutionizing orthopedic healthcare: a systematic review unveiling recombinant antimicrobial peptides.

The increasing demand for orthopedic surgeries, including joint replacements, is driven by an aging population and improved diagnosis of joint conditions. Orthopedic surgeries carry a risk of infection, especially in patients with comorbidities. The rise of antibiotic resistance exacerbates this issue, necessitating alternatives like in vitro bioengineered antimicrobial peptides (AMPs), offering broad-spectrum activity and multiple action mechanisms. This review aimed to assess the prevalence of antimicrobial potential and the yield after purification among recombinant AMP families. The antimicrobial potential was evaluated using the Minimum Inhibitory Concentration (MIC) values against the most common bacteria involved in clinical infections. This systematic review adhered to PRISMA guidelines, focusing on in vitro studies of recombinant AMPs. The search strategy was run on PubMed, Scopus and Embase up to 30th March 2023. The Population, Exposure and Outcome model was used to extract the data from studies and ToxRTool for the risk of bias analysis. This review included studies providing peptide production yield data and MIC values against pathogenic bacteria. Non-English texts, reviews, conference abstracts, books, studies focusing solely on chemical synthesis, those reporting incomplete data sets, using non-standard MIC assessment methods, or presenting MIC values as ranges rather than precise concentrations, were excluded. From 370 publications, 34 studies on AMPs were analyzed. These covered 46 AMPs across 18 families, with Defensins and Hepcidins being most common. Yields varied from 0.5 to 2,700 mg/L. AMPs were tested against 23 bacterial genera, with MIC values ranging from 0.125 to >1,152 µg/mL. Arenicins showed the highest antimicrobial activity, particularly against common orthopedic infection pathogens. However, AMP production yields varied and some AMPs demonstrated limited effectiveness against certain bacterial strains. This systematic review emphasizes the critical role of bioengineered AMPs to cope infections and antibiotic resistance. It meticulously evaluates recombinant AMPs, focusing on their antimicrobial efficacy and production yields. The review highlights that, despite the variability in AMP yields and effectiveness, Arenicins and Defensins are promising candidates for future research and clinical applications in treating antibiotic-resistant orthopedic infections. This study contributes significantly to the understanding of AMPs in healthcare, underscoring their potential in addressing the growing challenge of antibiotic resistance. Systematic review registration:https://osf.io/2uq4c/.

Cephalosporin C acylase: dream and(/or) reality.

Cephalosporins currently constitute the most widely prescribed class of antibiotics and are used to treat diseases caused by both Gram-positive and Gram-negative bacteria. Cephalosporins contain a 7-aminocephalosporanic acid (7-ACA) nucleus which is derived from cephalosporin C (CephC). The 7-ACA nucleus is not sufficiently potent for clinical use; however, a series of highly effective antibiotic agents could be produced by modifying the side chains linked to the 7-ACA nucleus. The industrial production of higher-generation semi-synthetic cephalosporins starts from 7-ACA, which is obtained by deacylation of the naturally occurring antibiotic CephC. CephC can be converted to 7-ACA either chemically or enzymatically using D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase. Both these methods show limitation, including the production of toxic waste products (chemical process) and the expense (the enzymatic one). In order to circumvent these problems, attempts have been undertaken to design a single-step means of enzymatically converting CephC to 7-ACA in the course of the past 10 years. The most suitable approach is represented by engineering the activity of a known glutaryl-7-aminocephalosporanic acid acylase such that it will bind and deacylate CephC more preferentially over glutaryl-7-aminocephalosporanic acid. Here, we describe the state of the art in the production of an effective and specific CephC acylase.

Correction to "Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid".

[This corrects the article DOI: 10.1021/acssuschemeng.2c06534.].