Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

Von-Hippel Lindau protein amyloid formation. The role of GST-tag.

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.

Production of native recombinant proteins using a novel split intein affinity technology.

Affinity tags are frequently engineered into recombinant proteins to facilitate purification. Although this technique is powerful, removal of the tag is desired because the tag can interfere with biological activity and can potentially increase the immunogenicity of therapeutic proteins. Tag removal is complex, as it requires adding expensive protease enzymes. To overcome this limitation, split intein based affinity purification systems have been developed in which a CC-intein tag is engineered into a protein of interest for binding to a NC-intein peptide ligand fixed to a chromatographic support. Tag removal in these systems is achieved by creating an active intein-complex during protein capture, which triggers a precise self-cleavage reaction. In this work, we show applications of a new split intein system, Cytiva™ ProteinSelect™. One advantage of the new system is that the NC-intein ligand can be robustly produced and conjugated to large volumes of resin for production of gram scale proteins. SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager in this work were successfully captured on the affinity resin and scaled 10-fold. Another advantage of this system is the ability to sanitize the resin with sodium hydroxide without loosing the 10-20 g/L binding capacity. Binding studies with IL-1b and IFNAR-1 ECD showed that the resin can be regenerated and sanitized for up to 50 cycles without loosing binding capacity. Additionally, after several cycles of sanitization, binding capacity was retained for the SARS-CoV-2 spike protein receptor binding domain and a Bispecific T Cell Engager. As with other split intein systems, optimization was needed to achieve ideal expression and recovery. The N-terminal amino acid sequence of the protein of interest required engineering to enable the cleavage reaction. Additionally, ensuring the stability of the CC-intein tag was important to prevent premature cleavage or truncation. Controlling the hold time of the expression product and the prevention of protease activity prior to purification was needed. These results demonstrate the feasibility of the Cytiva™ ProteinSelect™ system to be used in academic and industrial research and development laboratories for the purification of novel proteins expressed in either bacterial or mammalian systems.

Investigating the effect of SUMO fusion on solubility and stability of amylase-catalytic domain from Pyrococcus abyssi.

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of protein phase separation.

Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to ß-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

High-yield and cost-effective biosynthesis process for producing antimicrobial peptide AA139.

AA139, a variant of natural antimicrobial peptide (AMP) arenicin-3, displayed potent activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria. Nevertheless, there were currently few reports on the bioprocess of AA139, and the yields were less than 5 mg/L. Additionally, it was difficult and expensive to prepare AA139 through chemical synthesis due to its complex structure. These factors have impeded the further research and following clinical application of AA139. Here, we reported a bioprocess for the preparation of AA139, which was expressed in Escherichia coli (E. coli) BL21 (DE3) intracellularly in a soluble form via SUMO (small ubiquitin-related modifier) fusion technology. Then, recombinant AA139 (rAA139, refer to AA139 obtained by recombinant expression in this study) was obtained through the simplified downstream process, which was rationally designed in accordance with the physicochemical characteristics. Subsequently, the expression level of the interest protein was increased by 54% after optimization of high cell density fermentation (HCDF). Finally, we obtained a yield of 56 mg of rAA139 from 1 L culture with a purity of 98%, which represented the highest reported yield of AA139 to date. Furthermore, various characterizations were conducted to confirm the molecular mass, disulfide bonds, and antimicrobial activity of rAA139.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

"Mix and match" auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity.

Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Engineering Bacteriophytochrome-coupled Photoactivated Adenylyl Cyclases for Enhanced Optogenetic cAMP Modulation.

Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.

Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in Escherichia coli.

The strategy of active aggregation tag fusion expression with target proteins can solve the problems of restricted expression, inefficient purification, and laborious immobilization faced in the production of recombinant proteins in Escherichia coli. We localized a novel active aggregation peptide HlyA60 from the hemolysin A secretion system, which can effectively induce aggregate formation with satisfactory protein activities in E. coli after fusion expression with the protein of interest. Based on structural prediction and surface properties, the process of active aggregation of HlyA60 through electrostatic interactions and hydrophobic interactions was analyzed. To investigate the potential application of HlyA60 as an efficient aggregation tag, it was fused with acetyl xylan esterase and lipase A, separately. The resulting fusion proteins demonstrated active aggregation rates of 97.6 and 66.7%, respectively, leading to 1.9-fold and 1.7-fold increases in bacterial density at the end of fermentation. The AXE-HlyA60 fusion protein, which exhibited superior performance, was subjected to purification and immobilization. It was able to achieve column-free purification with an impressive 98.8% recovery and in situ immobilization; the immobilization enabled 30 cycles of reactions to take place with 85% residual activity maintained. Our findings provide a novel tool for efficiently producing recombinant proteins in E. coli.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action.

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.