Multiple Recombinant Proteins Research Articles

Open-source milligram-scale, four channel, automated protein purification system.

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.

A scalable synthesis of adjuvanting antigen depots based on metal-organic frameworks.

Vaccines have saved countless lives by preventing and even irradicating infectious diseases. Commonly used subunit vaccines comprising one or multiple recombinant proteins isolated from a pathogen demonstrate a better safety profile than live or attenuated vaccines. However, the immunogenicity of these vaccines is weak, and therefore, subunit vaccines require a series of doses to achieve sufficient immunity against the pathogen. Here, we show that the biomimetic mineralization of the inert model antigen, ovalbumin (OVA), in zeolitic imidazolate framework-8 (ZIF-8) significantly improves the humoral immune response over three bolus doses of OVA (OVA 3×). Encapsulation of OVA in ZIF-8 (OVA@ZIF) demonstrated higher serum antibody titers against OVA than OVA 3×. OVA@ZIF vaccinated mice displayed higher populations of germinal center (GC) B cells and IgG1+ GC B cells as opposed to OVA 3×, indicative of class-switching recombination. We show that the mechanism of this phenomenon is at least partly owed to the metalloimmunological effects of the zinc metal as well as the sustained release of OVA from the ZIF-8 composite. The system acts as an antigen reservoir for antigen-presenting cells to traffic into the draining lymph node, enhancing the humoral response. Lastly, our model system OVA@ZIF is produced quickly at the gram scale in a laboratory setting, sufficient for up to 20 000 vaccine doses.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Photoaffinity labelling displacement assay using multiple recombinant protein domains.

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR‑FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

Development of a novel serology assay for the detection of IgG antibodies to identify exposures to orthopoxviruses

Abstract The World Health Organization has declared a Public Health Emergency for an ongoing mpox (formerly monkeypox) outbreak, due to multiple cases of mpox in regions of the world in which infections have not been previously seen. The outbreak is notable for the high degree of human-human transmissions spreading primarily through sexual contacts. Currently, as a prophylactic (and post-exposure) measure, high-risk individuals are being offered immunization with cross-reactive vaccinia virus (VV) vaccines, a strategy previously used for the closely related variola (smallpox) virus. Commercial tests to measure binding antibodies to mpox for diagnostics or surveillance are not (yet) available. We present here a novel IgG detection assay to identify or measure binding antibodies to mpox and VV. This multiplexed, Luminex-based serology assay contains multiple recombinant proteins or peptides derived from VV and mpox. Because the assay contains antigens present on orthopoxviruses including VV, but also contain antigens present on orthopoxviruses exceptVV, the test allows for serological distinction between infection with virulent orthopoxviruses and attenuated strains of VV. We present data on detection of antibodies elicited by childhood (smallpox) or recent (mpox) vaccination, mpox infection, and breakthrough infection in previously vaccinated individuals. We demonstrate that the baseline antibody profiles of high-risk populations are distinct and must be considered when assessing mpox serology information. Overall, these data support the use of Luminex-based serology as a diagnostic tool for mpox infections and as a surveillance tool to detect the prevalence of mpox infections, even within a vaccinated population. This work was supported by Cooperative Agreement Number NU50CK000516 funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.

Recombinant Protein Vaccines Formulated with Enantio-Specific Cationic Lipid R-DOTAP Induce Protective Cellular and Antibody-Mediated Immune Responses in Mice.

Adjuvants are essential components of subunit vaccines added to enhance immune responses to antigens through immunomodulation. Very few adjuvants have been approved for human use by regulatory agencies due to safety concerns. Current subunit vaccine adjuvants approved for human use are very effective in promoting humoral immune responses but are less effective at promoting T-cell immunity. In this study, we evaluated a novel pure enantio-specific cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (R-DOTAP) as an immunomodulator for subunit vaccines capable of inducing both humoral- and cellular-mediated immunity. Using recombinant protein antigens derived from SARS-CoV2 spike or novel computationally optimized broadly reactive influenza antigen (COBRA) proteins, we demonstrated that R-DOTAP nanoparticles promoted strong cellular- and antibody-mediated immune responses in both monovalent and bivalent vaccines. R-DOTAP-based vaccines induced antigen-specific and polyfunctional CD8+ and CD4+ effector T cells and memory T cells, respectively. Antibody responses induced by R-DOTAP showed a balanced Th1/Th2 type immunity, neutralizing activity and protection of mice from challenge with live SARS-CoV2 or influenza viruses. R-DOTAP also facilitated significant dose sparing of the vaccine antigens. These studies demonstrate that R-DOTAP is an excellent immune stimulator for the production of next-generation subunit vaccines containing multiple recombinant proteins.

Steric accessibility of the N-terminus improves the titer and quality of recombinant proteins secreted from Komagataella phaffii

BackgroundKomagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly.ResultsWe determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein.ConclusionsOur observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.

Structural, functional, and immunogenicity implications of F9 gene recoding

AbstractHemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.

Nanoarchitectonics of a Microsphere-Based Scaffold for Modeling Neurodevelopment and Neurological Disease.

Three-dimensional cellular constructs derived from pluripotent stem cells allow the ex vivo study of neurodevelopment and neurological disease within a spatially organized model. However, the robustness and utility of three-dimensional models is impacted by tissue self-organization, size limitations, nutrient supply, and heterogeneity. In this work, we have utilized the principles of nanoarchitectonics to create a multifunctional polymer/bioceramic composite microsphere system for stem cell culture and differentiation in a chemically defined microenvironment. Microspheres could be customized to produce three-dimensional structures of defined size (ranging from >100 to <350 μm) with lower mechanical properties compared with a thin film. Furthermore, the microspheres softened in solution, approaching more tissue-like mechanical properties over time. With neural stem cells (NSCs) derived from human induced pluripotent stem cells, microsphere-cultured NSCs were able to utilize multiple substrates to promote cell adhesion and proliferation. Prolonged culture of NSC-bound microspheres under differentiating conditions allowed the formation of both neural and glial cell types from control and patient-derived stem cell models. Human NSCs and differentiated neurons could also be cocultured with astrocytes and human umbilical vein endothelial cells, demonstrating application for tissue-engineered modeling of development and human disease. We further demonstrated that microspheres allow the loading and sustained release of multiple recombinant proteins to support cellular maintenance and differentiation. While previous work has principally utilized self-organizing models or protein-rich hydrogels for neural culture, the three-dimensional matrix developed here through nanoarchitectonics represents a chemically defined and robust alternative for the in vitro study of neurodevelopment and nervous system disorders.

Comparison of novel recombinant nucleoprotein ELISA with commercial ELISA for Newcastle disease

In the present study, a novel ELISA method used recombinant nucleocapsid protein (rNP) as the coating agent. Recombinant Newcastle disease virus (NDV) protein was cloned and expressed in Escherichia coli. Though the rNP-ELISA results were consistent with commercial ELISA results for the NDV-negative sera samples, qualitatively and quantitatively variable (often reduced) results were obtained with NDV-positive sera. Although the rNP-ELISA results for NDV detection were inconclusive, further improvement and standardization of the rNP-ELISA approach, such as using multiple recombinant proteins as the ELISA coating agent and performing comprehensive statistical analyses of combined recombinant protein ELISA, should help counter Newcastle disease outbreaks by improving NDV detection.

Study on immunogenicity and antigenicity of a novel brucella multiepitope recombined protein

Currently, brucellosis is a reemerged zoonotic infectious disease with an increased incidence in recent years. A simple, rapid and sensitive method for diagnosing brucellosis can help to reduce medical burden and economic loss. Previously, a multiple epitope recombinant protein was constructed based on linear B-cell epitope prediction tools. In this study, the recombinant protein was used as an antigen to study the immune response produced by immunized mice, and goat serum was used to verify its diagnostic accuracy. The production of antibodies was successfully induced in the vaccinated mice. Flow cytometric analysis revealed that the percentage of CD4+, CD8+ and the CD4+/CD8+ ratios were increased by T cell subsets in mouse splenocytes, indicating that the recombinant protein induced a strong immune response had strong immunoreactivity. Using indirect ELISA, the recombinant protein correctly diagnosed positive and negative brucellosis samples. Compared with the whole bacterial antigen, the recombinant protein had a weaker sensitivity but a stronger specificity. Animal experiments showed that the recombinant protein had good antigenicity, and indirect ELISA indicates that it can be used as an antigen to diagnose brucellosis. Therefore, the recombinant protein is a potential candidate antigen for brucellosis vaccine development and serological diagnosis.

Navigating to the most promising directions amid complex fields of vaccine development: a chlamydial case study

ABSTRACTBackground: Vaccine-development research is proliferating making it difficult to determine the most promising vaccine candidates. Exemplary of this problem is vaccine development against Chlamydia, a pathogen of global public health and financial importance.Methods: We systematically extracted data from studies that included chlamydial load or host immune parameter measurements, estimating 4,453 standardized effect sizes between control and chlamydial immunization experimental groups.Results: Chlamydial immunization studies most often used (78%) laboratory mouse models. Depending on chlamydial species, single and multiple recombinant protein, viral and bacterial vectors, dendritic transfer, and dead whole pathogen were most effective at reducing chlamydial load. Immunization-driven decrease in chlamydial load was associated with increases in IFNg, IgA, IgG1, and IgG2a. Using data from individual studies, the magnitude of IgA and IgG2a increase was correlated with chlamydial load reduction. IFNg also showed this pattern for C. trachomatis, but not for C. muridarum. We also reveal the chlamydial vaccine development field to be highly bias toward studies showing these effects, limiting lessons learned from negative results.Conclusions: Most murine immunizations against Chlamydia reduced chlamydial load and increased host immune parameters. These methods are novel for vaccine development and are critical in identifying trends where large quantities of literature exist.

Characterization of two putative Dichelobacter nodosus footrot vaccine antigens identifies the first lysozyme inhibitor in the genus

The Gram-negative anaerobic bacterium Dichelobacter nodosus (Dn) causes footrot in ruminants, a debilitating and highly contagious disease that results in necrotic hooves and significant economic losses in agriculture. Vaccination with crude whole-cell vaccine mixed with multiple recombinant fimbrial proteins can provide protection during species-specific outbreaks, but subunit vaccines containing broadly cross-protective antigens are desirable. We have investigated two D. nodosus candidate vaccine antigens. Macrophage Infectivity Potentiator Dn-MIP (DNO_0012, DNO_RS00050) and Adhesin Complex Protein Dn-ACP (DNO_0725, DNO_RS06795) are highly conserved amongst ~170 D. nodosus isolates in the https://pubmlst.org/dnodosus/ database. We describe the presence of two homologous ACP domains in Dn-ACP with potent C-type lysozyme inhibitor function, and homology of Dn-MIP to other putative cell-surface and membrane-anchored MIP virulence factors. Immunization of mice with recombinant proteins with a variety of adjuvants induced antibodies that recognised both proteins in D. nodosus. Notably, immunization with fimbrial-whole-cell Footvax vaccine induced anti-Dn-ACP and anti-Dn-MIP antibodies. Although all adjuvants induced high titre antibody responses, only antisera to rDn-ACP-QuilA and rDn-ACP-Al(OH)3 significantly prevented rDn-ACP protein from inhibiting lysozyme activity in vitro. Therefore, a vaccine incorporating rDn-ACP in particular could contribute to protection by enabling normal innate immune lysozyme function to aid bacterial clearance.

Chitosan nano-structure loaded with recombinant E. coli O157:H7 antigens as a vaccine candidate can effectively increase immunization capacity

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

Abstract 5012: Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative

Abstract To meet the need for improved approaches to study cancer in vitro, there has been a surge in the development of novel research models utilizing advanced culture methods. These methods permit in vitro growth of cancer types previously not possible, and/or models with enhanced in vivo relevancy compared to traditional continuous cell lines. However, availability of these early-stage research models is currently limited and there is a lack of data on the ability to scale up production of these models to support the needs of the global research community. We sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort between the National Cancer Institute, the foundation Hubrecht Organoid Technology, Cancer Research UK, and the Wellcome Trust Sanger Institute. The HCMI's initial goal is the development of 1,000 novel human cancer models, paired with bioinformatics and patient clinical data, particularly from rare or underrepresented cancer types. One advanced culture method being utilized, three-dimensional organoid “microtissue” culture, potentially poses challenges for traditional large-scale bioproduction processes. It requires growth embedded within an undefined extracellular matrix and complex media formulations containing multiple small molecules and recombinant proteins with unknown stability and shelf-life. Additionally, organoid growth media typically includes multiple sources of undefined conditioned media containing critical growth factors. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. Multiple unique donors were available for all tissues and both cancer and non-cancer models were available for two tissue types. Most models were maintained in culture continuously for at least 60 days (7-27 population doublings, &amp;gt; 10 passages). Tissue and donor variability was evident in model characteristics, including morphology (assessed by microscopy and immunocytochemistry), growth rate, and genetic stability (measured by short tandem repeats analysis). All models were amenable to scale up beyond multiwell plates, and all models could recover from cryopreservation. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research. Citation Format: James Clinton, Penney McWilliams-Koeppen, Siddhartha Paul, Allison Ruchinskas, Dezhong Yin, Robert Newman. Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5012.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3 ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3 ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3 ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3 ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3 ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3 ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3 ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3 ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3 ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound

Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.

Vaccine self-assembling immune matrix is a non-viral delivery platform that improves overall vaccine performance while maintaining safety and stability

Abstract Vaccination remains the most effective public health tool to prevent infectious disease. Many vaccines are marginally effective, and are less so when administered to immune-compromised populations. To enhance vaccine immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create a new vaccine delivery method, VacSIM® (vaccine self-assembling immune matrix). Vaccine components are mixed with the VacSIM® solution for injection, after which the (RADA)4 peptides self-assemble into hydrated nanofiber gel-matrices, forming a vaccine depot with the vaccine antigens in the aqueous phase, allowing for slow-release of vaccine components over time. Thus, we have a non-viral, inert, biodegradable delivery system, not requiring formulation that can deliver various types of vaccines. We hypothesize that slow-release of vaccine components provides antigen persistence, driving enhanced vaccine-specific responses that are improved in both quantity and quality. We have tested VacSIM® with live bacterial vectors, inactivated virus and multiple recombinant protein vaccines. VacSIM® delivery of different protein immunogens, including Hepatitis B and HIV Envelope, is superior to delivery in licensed and unlicensed adjuvants and requires only a single injection. VacSIM® improves existing vaccines by reducing the number of injections needed, improving vaccine performance, especially in difficult-to-protect populations like the elderly and immune compromised, and reducing unwelcome side effects. VacSIM® can be stockpiled and stored without a cold chain, which is especially beneficial in resource-limited settings where vaccine access and compliance are issues.

125 years of virology and ascent of biotechnologies based on viral expressio

The study of viruses lasts for more than a century since their discovery in 1892. In recent decades, viruses are also being actively exploited as a biotechnological tool. Plant-virus-driven transient expression of heterologous proteins is an actively developing production platform; it is the basis of several industrial processes that are currently being used for the production of multiple recombinant proteins. Viral vectors have also become useful tools for research. Viral vectors delivered by Agrobacterium (magnifection) provide for high protein yield, rapid scale up and fast manufacturing. In this review, we explore modern approaches for biotechnological production of recombinant proteins in plants using viral vectors.

A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression. IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2AE expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2AE system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii.