Recombinant Protein Expression Research Articles

Salmonella-Delivered COBRA-HA1 Antigen Derived from H1N1 Hemagglutinin Sequences Elicits Broad-Spectrum Protection Against Influenza A Subtypes

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4+ and CD8+ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4+ and CD8+ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.

Elucidation of non-catalytic domain alterations effects on properties and action pattern of a novel alginate lyase

The discovery and characterization of new alginate lyases with high activity and stability have been the research topic in utilization of alginate biomass. Herein, we characterized a new Thalassotalea algicola-derived polyM-preferred PL6 family alginate lyase TAPL6 with two-domains (catalytic domain NTD and non-catalytic domain CTD). TAPL6 is a bifunctional enzyme that could endolytically degrade polyM, and could exolytically degrade polyG. It could degrade alginate in a hybrid action mode. The specific activities of polyM, polyG and sodium alginate of TAPL6 were 7174 U/mg, 4140.2 U/mg and 414.3 U/mg, respectively. The optimal temperature and pH for TAPL6 are 25 °C and 8.0, respectively. Deletion of CTD resulted in a loss of 99.8%− 98.2% of TAPL6 activity and a decrease in pH adaptability, but greatly improved the activity and temperature stability above 30 °C. The CTD truncation is more resistant to metal ions than TAPL6, and the promotion effect of Ca2+ on the activity is stronger. Deletion of CTD results in a change in the action mode of TAPL6 to an endo-mode. In addition, the integrity of the linker between NTD and CTD is very important for the soluble expression of recombinant protein, and the complete linker is beneficial to the catalytic activity of NTD. This study provides a new tool enzyme for the production of alginate oligosaccharides, and explores the role of the non-catalytic domain CTD in the PL6 family alginate lyases, providing a rational design basis for the PL6 family alginate lyases to change the enzymatic properties and action mode.

Secretion-Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface.

Protein-based biomaterials have played a key role in tissue engineering, and additional exciting applications as self-healing materials and sustainable polymers are emerging. Over the past few decades, recombinant expression and production of various fibrous proteins from microbes have been demonstrated; however, the resulting proteins typically must then be purified and processed by humans to form usable fibers and materials. Here, we show that the Gram-positive bacterium Bacillus subtilis can be programmed to secrete silk through its translocon via an orthogonal signal peptide/peptidase pair. Surprisingly, we discover that this translocation mechanism drives the silk proteins to assemble into fibers spontaneously on the cell surface, in a process we call secretion-catalyzed assembly (SCA). Secreted silk fibers form self-healing hydrogels with minimal processing. Alternatively, the fibers retained on the membrane provide a facile route to create engineered living materials from Bacillus cells. This work provides a blueprint to achieve autonomous assembly of protein biomaterials in useful morphologies directly from microbial factories.

Secretion‐Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface

AbstractProtein‐based biomaterials have played a key role in tissue engineering, and additional exciting applications as self‐healing materials and sustainable polymers are emerging. Over the past few decades, recombinant expression and production of various fibrous proteins from microbes have been demonstrated; however, the resulting proteins typically must then be purified and processed by humans to form usable fibers and materials. Here, we show that the Gram‐positive bacterium Bacillus subtilis can be programmed to secrete silk through its translocon via an orthogonal signal peptide/peptidase pair. Surprisingly, we discover that this translocation mechanism drives the silk proteins to assemble into fibers spontaneously on the cell surface, in a process we call secretion‐catalyzed assembly (SCA). Secreted silk fibers form self‐healing hydrogels with minimal processing. Alternatively, the fibers retained on the membrane provide a facile route to create engineered living materials from Bacillus cells. This work provides a blueprint to achieve autonomous assembly of protein biomaterials in useful morphologies directly from microbial factories.

SCREENING OF VARIOUS PROMOTERS FOR INCREASED PROTEIN EXPRESSION

One of the essential elements in the in expression of recombinant proteins in Eshecichia coli is a promoter. It is a sequence of DNA that drives the expression of recombinant protein production. Along with the choice of promoter a number of factors such as protein parameters, induction temperature, inducer, induction conditions and protein solubility play a major role in deciding the yield of target protein. In the present study, we have evaluated the strength of three different promoters (phoA, cstA and pLpR) and compared it with one of the strong promoters known, T7. A vector with these three promoters were constructed for expression of FCTRX 1 – 15 tagged proinsulin gene. Protein expressed was isolated, solubilized and purified using chromatography. Yields of proinsulin obtained from different promoter driven constructs was compared and it was observed that the inclusion bodies (IB) yield obtained from temperature inducible pLpR system was comparable with T7 system and this can be further increased by optimizing the fermentation conditions.

Expression and Purification of Recombinant T7 RNA Polymerase in Escherichia Coli

T7 RNA polymerase, DNA dependent RNA polymerase commonly employed in virus molecular research, in vitro transcription and virus replication. The aim of this study was to produce a recombinant T7 RNA polymerase enzyme employing an E. coli protein expression system. In addition, this project aimed to establish and optimize high-yield expression of soluble recombinant T7 RNA polymerase in E. coli cells. Escherichia coli are the most commonly used protein expression systems because of the ease of cultivation, high yield, and low cost. E. coli expression system based on T7 promoter (pRSET-a) was used for T7 RNA polymerase recombinant protein expression. The T7 RNAP gene was amplified and ligated into a pRSET-an expression vector with a hexa-histidine tag at the N-terminus. The ligated vector was effectively transformed into E. coli BL21 (DE3). For T7 RNAP recombinant protein overexpression, it was confirmed that T7 RNA polymerase gene could be induced with IPTG up to 1.0mM, when the OD600 of the culture broth reached 0.5-1.2. The recombinant T7 RNA polymerase was harvested from the whole cell lysate after three hours of induction at 18oC. T7 RNA polymerase recombinant protein was efficiently purified by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. SDS-PAGE and western blot analysis confirmed the identity of the purified T7 RNA polymerase recombinant protein in soluble fractions with purity in the range of 60%. T7 RNA polymerase recombinant protein expression can be achieved by considering specific factors and suitable conditions.

Time-Course Transcriptome Analysis of Bacillus subtilis DB104 during Growth.

Bacillus subtilis DB104, an extracellular protease-deficient derivative of B. subtilis 168, is widely used for recombinant protein expression. An understanding of the changes in gene expression during growth is essential for the commercial use of bacterial strains. Transcriptome and proteome analyses are ideal methods to study the genomic response of microorganisms. In this study, transcriptome analysis was performed to monitor changes in the gene expression level of B. subtilis DB104 while growing on a complete medium. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, K-mean cluster analysis, gene ontology (GO) enrichment analysis, and the function of sigma factors were used to divide 2122 differentially expressed genes (DEGs) into 10 clusters and identified gene functions according to expression patterns. The results of KEGG pathway analysis indicated that ABC transporter is down-regulated during exponential growth and metabolic changes occur at the transition point where sporulation starts. At this point, several stress response genes were also turned on. The genes involved in the lipid catabolic process were up-regulated briefly at 15 h as an outcome of the programmed cell death that postpones sporulation. The results suggest that changes in the gene expression of B. subtilis DB104 were dependent on the initiation of sporulation. However, the expression timing of the spore coat gene was only affected by the relevant sigma factor. This study can help to understand gene expression and regulatory mechanisms in B. subtilis species by providing an overall view of transcriptional changes during the growth of B. subtilis DB104.

Directed evolution of Mesorhizobium loti carbonic anhydrase for carbon dioxide sequestration by MutaT7 and rational codon design

BackgroundDirected evolution plays a crucial role in accelerating genetic development to obtain superior enzyme variants with high tolerance and activity. Under nature evolution, Mesorhizobium loti carbonic anhydrase (MlCA) stands out as one of the fastest metalloenzymes involved in carbon dioxide capture and sequestration (CCS). However, challenge arises when expressing the recombinant MlCA which affects its total catalytic function. Therefore, we aim to evolve MlCA by enhancing protein production and elevate catalytic efficiency in genetically Escherichia coli. MethodsA growth-dependent CRISPRi-based high-throughput screening platform DEPEND-2.0 with down-regulating essential gene was established. The in vivo target-specific MutaT7 mutator was employed to evolve MlCA and screened the mutants using DEPEND-2.0 based on cell growth. Structure prediction was further applied to analyze the mutant while the degenerated or definitive codons were designed to investigate the codon influence on the mRNA and enzyme production. Significant findingThe DEPEND-2.0 system, utilizing chromosomal dCas9, effectively inhibited cell growth by targeting the promoter region with sgRNA which maintained a repression rate of 65.4% at 4 h using high-copy RSF plasmid. Structural prediction using Alphafold revealed that a mutation introduced by MutaT7 at P105S resulted in a 99% loss of intrinsic activity due to the disruption of hydrogen bonding. Furthermore, synonymous codon designs demonstrated that AT-rich codons at the 5’ terminus of a 30-bp sequence exhibited higher mRNA levels and improved recombinant protein expression. Finally, the MlCA variant with A-rich codons (i.e., MA) effectively facilitated CO2 biomineralization, displaying high durability with 72% residual activity after five repeated reactions, ultimately accumulating 193 mg of CaCO3.

Protein-Specific Signal Peptides for Mammalian Vector Engineering.

Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese hamster ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive endoplasmic reticulum (ER) translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination of in silico and targeted design rule-based in vitro testing can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers ≥1.8×, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlined in silico/in vitro testing processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Lactiplantibacillus plantarum enables blood urate control in mice through degradation of nucleosides in gastrointestinal tract

BackgroundLactobacillus species in gut microbiota shows great promise in alleviation of metabolic diseases. However, little is known about the molecular mechanism of how Lactobacillus interacts with metabolites in circulation. Here, using high nucleoside intake to induce hyperuricemia in mice, we investigated the improvement in systemic urate metabolism by oral administration of L. plantarum via different host pathways.ResultsGene expression analysis demonstrated that L. plantarum inhibited the activity of xanthine oxidase and purine nucleoside phosphorylase in liver to suppress urate synthesis. The gut microbiota composition did not dramatically change by oral administration of L. plantarum over 14 days, indicated by no significant difference in α and β diversities. However, multi-omic network analysis revealed that increase of L. plantarum and decrease of L. johnsonii contributed to a decrease in serum urate levels. Besides, genomic analysis and recombinant protein expression showed that three ribonucleoside hydrolases, RihA–C, in L. plantarum rapidly and cooperatively catalyzed the hydrolysis of nucleosides into nucleobases. Furthermore, the absorption of nucleobase by intestinal epithelial cells was less than that of nucleoside, which resulted in a reduction of urate generation, evidenced by the phenomenon that mice fed with nucleobase diet generated less serum urate than those fed with nucleoside diet over a period of 9-day gavage.ConclusionCollectively, our work provides substantial evidence identifying the specific role of L. plantarum in improvement of urate circulation. We highlight the importance of the enzymes RihA–C existing in L. plantarum for the urate metabolism in hyperuricemia mice induced by a high-nucleoside diet. Although the direct connection between nucleobase transport and host urate levels has not been identified, the lack of nucleobase transporter in intestinal epithelial cells might be important to decrease its absorption and metabolization for urate production, leading to the decrease of serum urate in host. These findings provide important insights into urate metabolism regulation.BbgstkDZoHsJm_QsUh-oZNVideo

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Effects of signal peptides on the secreted green fluorescent protein (GFP) expression by recombinant Saccharomyces cerevisiae

Recombinant proteins secreted by Saccharomyces cerevisiae have been widely applied in the food industry and biopharmaceuticals because of their valuable features. However, low-secreted protein production in S. cerevisiae is a pending challenge that has hampered applications based on S. cerevisiae. To improve the secreted protein expression in S. cerevisiae, it is important to develop an efficient cassette for the secretory expression of recombinant protein by S. cerevisiae. In this study, three cassettes were constructed in which a gene of interest was fused with genes coding for secretory signal peptides from AGA2, MFα and α-glucoamylase. Green fluorescent protein (GFP) was employed as a model protein to investigate the secretion of recombinant protein mediated by these signal peptides in S. cerevisiae. The secreted expression of GFP was detected by Western Blot and dot-blot analyses using a specific antibody against GFP which showed the strongest GFP levels from MFα signal-related cassette. Therefore, their cassettes could be employed for following studies on the secretion of some other proteins.

Construction and expression of Wee1 recombinant protein in Escherichia coli strain BL21 (DE3)

Wee1 is a gene encoding for protein kinase that is located in the nucleus and it plays an essential role in determining the timing of mitosis. Overexpression of Wee1 in rice is resulting in increased plant size. However, the increasing plant size due to the increase of Wee1 protein expression is not elucidated. Immunodiagnostic using specific antibodies against Wee1 protein should be conducted to determine the role of the protein on cell division. This experiment was directed to construct Wee1 in an expression vector for synthesis of recombinant Wee1 protein using three strategies of construction. The three strategies were construction of full length (FL), deletion of putative binding site (BS), and deletion of N-terminal domain (ΔN) of Wee1. Restriction analysis with BamH1/Sal1 of FL-Wee1, BS-Wee1, and ΔN-Wee1 construct resulted in the different DNA fragments with molecular size at 1239, 1176, and 960bp, respectively. This result indicated that the Wee1 fragments have been successfully inserted in the expression plasmid which was further confirmed using DNA sequencing. Colony PCR analysis showed that Escherichia coli strain BL21 (DE3) has been transformed with the constructs. The protein analysis using SDS-PAGE revealed that the recombinant protein of Wee1 synthesized in the E.coli containing ΔN-Wee1 construct, but not in FL-Wee1 and BS-Wee1 constructs. The ΔN-Wee1 protein was synthesized in an insoluble fraction with a molecular size of 38.8 kDa which is the same as the size estimated using the software ExPASy. Interestingly, the level of synthesized ΔN-Wee1 protein was not induced by IPTG concentration. Collectively, the results indicated that the DNA construct of ΔN-Wee1 is suitable for recombinant protein production.

Immunogenicity Analysis of PCV3 Recombinant Capsid Protein Virus-like Particles and Their Application in Antibodies Detection.

Porcine circovirus type 3 is a newly emerging pathogen of porcine circovirus associated disease (PCVAD). Currently, there is no commercially available vaccine, resulting in huge economic losses to the pig industry. Porcine circovirus type 3 capsid protein (Cap) can self-assemble into virus-like particles (VLPs). Therefore, the expression of the recombinant Cap protein is of great significance for the prevention, diagnosis and control of porcine circovirus type 3 associated diseases. In this study, the recombinant Cap protein was successfully expressed in Escherichia coli by deleting the nuclear localization sequence (NLS). The VLPs were observed by transmission electron microscopy. To evaluate the immunogenicity of the recombinant Cap protein, mice were immunized. As a result, the recombinant Cap protein can induce higher levels of humoral and cellular immune responses. A VLP-based ELISA method was developed for the detection of antibodies. The established ELISA method has good sensitivity, specificity, repeatability and clinical applicability. These results demonstrate the successful expression of the PCV3 recombinant Cap protein and the preparation of recombinant Cap protein VLPs, which can be used for the preparation of subunit vaccines. Meanwhile, the established I-ELISA method lays a foundation for the development of the commercial PCV3 serological antibody detection kit.

Risk assessment and bioburden evaluation of Agrobacterium tumefaciens-mediated transient protein expression in plants using the CaMV35S promoter

Large-scale transient expression of recombinant proteins in plants is increasingly used and requires the multi-liter cultivation of Agrobacterium tumefaciens transformed with an expression vector, which is often cloned in Escherichia coli first. Depending on the promoter, unintentional activity can occur in both bacteria, which could pose a safety risk to the environment and operators if the protein is toxic. To assess the risk associated with transient expression, we first tested expression vectors containing the CaMV35S promoter known to be active in plants and bacteria, along with controls to measure the accumulation of the corresponding recombinant proteins. We found that, in both bacteria, even the stable model protein DsRed accumulated at levels near the detection limit of the sandwich ELISA (3.8 µg L−1). Higher levels were detected in short cultivations (< 12 h) but never exceeded 10 µg L−1. We determined the abundance of A. tumefaciens throughout the process, including infiltration. We detected few bacteria in the clarified extract and found none after blanching. Finally, we combined protein accumulation and bacterial abundance data with the known effects of toxic proteins to estimate critical exposures for operators. We found that unintended toxin production in bacteria is negligible. Furthermore, the intravenous uptake of multiple milliliters of fermentation broth or infiltration suspension would be required to reach acute toxicity even when handling the most toxic products (LD50 ~ 1 ng kg−1). The unintentional uptake of such quantities is unlikely and we therefore regard transient expression as safe in terms of the bacterial handling procedure.

Protein production from HEK293 cell line-derived stable pools with high protein quality and quantity to support discovery research.

Antibody-based therapeutics and recombinant protein reagents are often produced in mammalian expression systems, which provide human-like post-translational modifications. Among the available mammalian cell lines used for recombinant protein expression, Chinese hamster ovary (CHO)-derived suspension cells are generally utilized because they are easy to culture and tend to produce proteins in high yield. However, some proteins purified from CHO cell overexpression suffer from clipping and display undesired non-human post translational modifications (PTMs). In addition, CHO cell lines are often not suitable for producing proteins with many glycosylation motifs for structural biology studies, as N-linked glycosylation of proteins poses challenges for structure determination by X-ray crystallography. Hence, alternative and complementary cell lines are required to address these issues. Here, we present a robust method for expressing proteins in human embryonic kidney 293 (HEK293)-derived stable pools, leading to recombinant protein products with much less clipped species compared to those expressed in CHO cells and with higher yield compared to those expressed in transiently-transfected HEK293 cells. Importantly, the stable pool generation protocol is also applicable to HEK293S GnTI- (N-acetylglucosaminyltransferase I-negative) and Expi293F GnTI- suspension cells, facilitating production of high yields of proteins with less complex glycans for use in structural biology projects. Compared to HEK293S GnTI- stable pools, Expi293F GnTI- stable pools consistently produce proteins with similar or higher expression levels. HEK293-derived stable pools can lead to a significant cost reduction and greatly promote the production of high-quality proteins for diverse research projects.

Application of immuno- and affinity labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells

In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λem ∼550 nm) and far-red (λem ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.

Design principles and applications of synthetic self-replicating RNAs.

With the advent of ever more sophisticated methods for the in vitro synthesis and the in vivo delivery of RNAs, synthetic mRNAs have gained substantial interest both for medical applications, as well as for biotechnology. However, in most biological systems exogeneous mRNAs possess only a limited half-life, especially in fast dividing cells. In contrast, viral RNAs can extend their lifetime by actively replicating inside their host. As such they may serve as scaffolds for the design of synthetic self-replicating RNAs (srRNA), which can be used to increase both the half-life and intracellular concentration of coding RNAs. Synthetic srRNAs may be used to enhance recombinant protein expression or induce the reprogramming of differentiated cells into pluripotent stem cells but also to create cell-free systems for research based on experimental evolution. In this article, we discuss the applications and design principles of srRNAs used for cellular reprogramming, mRNA-based vaccines and tools for synthetic biology. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Refinement of the Fusion Tag PagP for Effective Formation of Inclusion Bodies in Escherichia coli.

Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation "hot spots" (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the β-strands. Substitution of these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable β-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.