Factors affecting therapeutic protein purity and yield during chromatographic purification.

Therapeutic proteins are recombinant proteins generated through recombinant DNA technology and have attracted a great deal of interest in numerous applications, including pharmaceutical, cosmetic, human and animal health, agriculture, food, and bioremediation. Producing therapeutic proteins on a large scale, mainly in the pharmaceutical industry, necessitates a cost-effective, straightforward, and adequate manufacturing process. In industry, a protein separation technique based mainly on protein characteristics and modes of chromatography will be applied to optimize the purification process. Typically, the downstream process of biopharmaceutical operations may involve multiple chromatography phases that require the use of large columns pre-packed with resins that must be inspected before use. Approximately 20% of the proteins are assumed to be lost at each purification stage during the production of biotherapeutic products. Hence, to produce a high quality product, particularly in the pharmaceutical industry, the correct approach and understanding of the factors influencing purity and yield during purification are necessary.

GEnZ explorer: a tool for visualizing agroclimate to inform research and regulatory risk assessment.

Confined field trials (CFT) of genetically engineered (GE) crops are used to generate data to inform environmental risk assessments (ERA). ERAs are required by regulatory authorities before novel GE crops can be released for cultivation. The transportability of CFT data to inform risk assessment in countries other than those where the CFT was conducted has been discussed previously in an analysis showing that the primary difference between CFT locations potentially impacting trial outcomes is the physical environment, particularly the agroclimate. This means that data from trials carried out in similar agroclimates could be considered relevant and sufficient to satisfy regulatory requirements for CFT data, irrespective of the country where the CFTs are conducted. This paper describes the development of an open-source tool to assist in determining the transportability of CFT data. This tool provides agroclimate together with overall crop production information to assist regulators and applicants in making informed choices on whether data from previous CFTs can inform an environmental risk assessment in a new country, as well as help developers determine optimal locations for planning future CFTs. The GEnZ Explorer is a freely available, thoroughly documented, and open-source tool that allows users to identify the agroclimate zones that are relevant for the production of 21 major crops and crop categories or to determine the agroclimatic zone at a specific location. This tool will help provide additional scientific justification for CFT data transportability, along with spatial visualization, to help ensure regulatory transparency.

Manufacturing innovation to drive down cell therapy costs.

Chimeric antigen receptor T cells (CAR-T) have demonstrated their potential to revolutionize cancer treatment. However, manufacturing remains a challenge. Multiple manufacturing innovations [e.g., vector and gene engineering, process improvements, hardware innovation, digital innovation, and point-of-care (POC) manufacturing] have the potential to help realize the full potential of CAR-T therapies.

Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application.

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at -20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3'-5' exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.

Recent advances in genetic technology development of oleaginous yeasts.

As important chemical raw materials and potential nutritional supplements, microbial lipids play an important role in ensuring economic development, food security, and energy security. Compared with non-natural hosts, oleaginous yeasts exhibit obvious advantages in lipid yield and productivity and have great potential to be genetically engineered into an oil cell factory. The main bottleneck in the current oleaginous yeasts engineering is the lack of genetic manipulation tools. Fortunately, the rapid development of synthetic biology has provided numerous new approaches, resources, and ideas for the field. Most importantly, gene editing technology mediated by CRISPR/Cas systems has been successfully applied to some oleaginous yeasts, almost completely rewriting the development pattern of genetic manipulation technology applicable. This paper reviews recent progress in genetic technology with regard to oleaginous yeasts, with a special focus on transformation methods and genome editing tools, discussing the effects of some important genetic parts. KEY POINTS: •Contribution of microbiotechnology in food safety and biofuel by oleaginous yeasts. •Advancement of genetic manipulation and transformation for oleaginous yeasts.

Genetic modifications associated with sustainability aspects for sustainable developments.

Sustainable development serves as the foundation for a range of international and national policymaking. Traditional breeding methods have been used to modify plant genomes and production. Genetic engineering is the practice of assisting agricultural systems in adapting to rapidly changing global growth by hastening the breeding of new varieties. On the other hand, the development of genetic engineering has enabled more precise control over the genomic alterations made in recent decades. Genetic changes from one species can now be introduced into a completely unrelated species, increasing agricultural output or making certain elements easier to manufacture. Harvest plants and soil microorganisms are just a few of the more well-known genetically modified creatures. Researchers assess current studies and illustrate the possibility of genetically modified organisms (GMOs) from the perspectives of various stakeholders. GMOs increase yields, reduce costs, and reduce agriculture's terrestrial and ecological footprint. Modern technology benefits innovators, farmers, and consumers alike. Agricultural biotechnology has numerous applications, each with its own set of potential consequences. This will be able to reach its full potential if more people have access to technology and excessive regulation is avoided. This paper covers the regulations for genetically modified crops (GMCs) as well as the economic implications. It also includes sections on biodiversity and environmental impact, as well as GMCs applications. This recounts biotechnological interventions for long-term sustainability in the field of GMCs, as well as the challenges and opportunities in this field of research.Abbreviations: GMCs-Genetically modified crops; GMOs- Genetically modified organisms; GE- Genetic engineering; Bt- Bacillus thuringiensisNIH- National Institutes of Health; FDA- Food and Drug Administration; HGT- Horizontal gene transfer; GM- Genetically modified; rDNA- Ribosomal deoxyribonucleic acid; USDA- United States Department of Agriculture; NIH- National Institutes of Health.

Removal of toxic heavy metals using genetically engineered microbes: Molecular tools, risk assessment and management strategies.

The direct release of industrial effluent into the water and other anthropogenic activities causes water pollution. Heavy metal ions are the primary contaminant in the industrial effluents which are exceptionally toxic at low concentrations, terribly disturb the endurance equilibrium of activities in the eco-system and be remarkably hazardous to human health. Different conventional treatment methodologies were utilized for the removal of toxic pollutants from the contaminated water which has several drawbacks such as cost-ineffective and lower efficiency. Recently, genetically modified micro-organisms (GMMs) stand-out for the removal of toxic heavy metals are viewed as an economically plausible and environmentally safe technique. GMMs are microorganisms whose genetic material has been changed utilizing genetic engineering techniques that exhibit enhanced removal efficiency in comparison with the other treatment methodologies. The present review comments the GMMs such as bacteria, algae and fungi and their potential for the removal of toxic heavy metals. This review provides current aspects of different advanced molecular tools which have been used to manipulate micro-organisms through genetic expression for the breakdown of metal compounds in polluted areas. The strategies, major limitations and challenges for genetic engineering of micro-organisms have been reviewed. The current review investigates the approaches working on utilizing genetically modified micro-organisms and effective removal techniques.

iGEM and Gene Drives: A Case Study for Governance.

Gene drives have already challenged governance systems. In this case study, we explore the International Genetically Engineered Machine (iGEM) competition's experiences in gene drive-related research and lessons in developing, revising, and implementing a governance system. iGEM's experiences and lessons are distilled into 6 key insights for future gene drive policy development in the United States: (1) gene drives deserve special attention because of their potential for widescale impact and remaining uncertainty about how to evaluate intergenerational and transboundary risks; (2) an adaptive risk management approach is logical for gene drives because of the rapidly changing technical environment; (3) review by individual technical experts is limited and may fail to incorporate other forms of expertise and, therefore, must be complemented with a range of alternative governance methods; (4) current laboratory biosafety and biosecurity review processes may not capture gene drive research or its components in practice even if they are covered theoretically; (5) risk management for research and development must incorporate discussions of values and broader implications of the work; and (6) a regular technology horizon scanning capacity is needed for the early identification of advances that could pose governance system challenges.

Development and evaluation of a low cost IgG ELISA test based in RBD protein for COVID-19.

Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.

Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability.

Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12-302 ss] was not further active which could be due to the large truncation. However, T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25-90 °C and pH 6-9.5. Interestingly, remaining enzyme activity of T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8-339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry.

For Faster Science: Accelerated Genetic Engineering.

While acknowledging that there are many risks associated with genetic engineering, this article asserts that delaying the research and development of genetic engineering has high human costs. Genetic engineering could prevent millions of premature deaths, eliminate the suffering associated with many diseases and conditions, and save millions of family members from the anguish of watching their loved ones suffer and die from genetic conditions. Societal deliberations on the topic of genetic engineering have existed for decades, and a majority of people now support genetic interventions. Highly legitimate social justice issues can be addressed without holding advances in science hostage.

Biotechnological Production of Carotenoids Using Low Cost-Substrates Is Influenced by Cultivation Parameters: A Review.

Carotenoids are natural lipophilic pigments mainly found in plants, but also found in some animals and can be synthesized by fungi, some bacteria, algae, and aphids. These pigments are used in food industries as natural replacements for artificial colors. Carotenoids are also known for their benefits to human health as antioxidants and some compounds have provitamin A activity. The production of carotenoids by biotechnological approaches might exceed yields obtained by extraction from plants or chemical synthesis. Many microorganisms are carotenoid producers; however, not all are industrially feasible. Therefore, in this review, we provide an overview regarding fungi that are potentially interesting to industry because of their capacity to produce carotenoids in response to stresses on the cultivation medium, focusing on low-cost substrates.

Genetically engineered microbial remediation of soils co-contaminated by heavy metals and polycyclic aromatic hydrocarbons: Advances and ecological risk assessment.

Soils contaminated with heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) have been becoming a worldwide concerned environmental problem because of threatening public healthy via food chain exposure. Thus soils polluted by HMs and PAHs need to be remediated urgently. Physical and chemical remediation methods usually have some disadvantages, e.g., cost-expensiveness and incomplete removal, easily causing secondary pollution, which are hence not environmental-friendly. Conventional microbial approaches are mostly used to treat a single contaminant in soils and lack high efficiency and specificity for combined contaminants. Genetically engineered microorganisms (GEMs) have emerged as a desired requirement of higher bioremediation efficiency for soils polluted with HMs and PAHs and environmental sustainability, which can provide a more eco-friendly and cost-effective strategy in comparison with some conventional techniques. This review comments the recent advances about successful bioremediation techniques and approaches for soil contaminated with HMs and/or PAHs by GEMs, and discusses some challenges in the simultaneous removal of HMs and PAHs from soil by designing multi-functional genetic engineering microorganisms (MFGEMs), such as improvement of higher efficiency, strict environmental conditions, and possible ecological risks. Also, the modern biotechnological techniques and approaches in improving the ability of microbial enzymes to effectively degrade combined contaminants at a faster rate are introduced, such as reasonable gene editing, metabolic pathway modification, and protoplast fusion. Although MFGEMs are more potent than the native microbes and can quickly adapt to combined contaminants in soils, the ecological risk of MFGEMs needs to be evaluated under a regulatory, safety, or costs benefit-driving system in a way of stratified regulation. Nevertheless, the innovation of genetic engineering to produce MFGEMs should be inspired for the welfare of successful bioremediation for soils contaminated with HMs and PAHs but it must be supervised by the public, authorities, and laws.

A deterministic genotyping workflow reduces waste of transgenic individuals by two-thirds.

We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.

Genetically modified crop regulations: scope and opportunity using the CRISPR-Cas9 genome editing approach.

Global demand for food is increasing day by day due to an increase in population and shrinkage of the arable land area. To meet this increasing demand, there is a need to develop high-yielding varieties that are nutritionally enriched and tolerant against environmental stresses. Various techniques are developed for improving crop quality such as mutagenesis, intergeneric crosses, and translocation breeding. Later, with the development of genetic engineering, genetically modified crops came up with the transgene insertion approach which helps to withstand adverse conditions. The process or product-focused approaches are used for regulating genetically modified crops with their risk analysis on the environment and public health. However, recent advances in gene-editing technologies have led to a new era of plant breeding by developing techniques including site-directed nucleases, zinc finger nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) that involve precise gene editing without the transfer of foreign genes. But these techniques always remain in debate for their regulation status and public acceptance. The European countries and New Zealand, consider the gene-edited plants under the category of genetically modified organism (GMO) regulation while the USA frees the gene-edited plants from such type of regulations. Considering them under the category of GMO makes a long and complicated approval process to use them, which would decrease their immediate commercial value. There is a need to develop strong regulatory approaches for emerging technologies that expedite crop research and attract people to adopt these new varieties without hesitation.

Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables.

The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4-5 d.

Non-invasive assessment of telomere maintenance mechanisms in brain tumors.

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.

CRISPRing future medicines.

Introduction: The ability to engineer mammalian genomes in a quick and cost-effective way has led to rapid adaptation of CRISPR technology in biomedical research. CRISPR-based engineering has the potential to accelerate drug discovery, to support the reduction of high attrition rate in drug development and to enhance development of cell and gene-based therapies.Areas covered: How CRISPR technology is transforming drug discovery is discussed in this review. From target identification to target validation in both in vitro and in vivo models, CRISPR technology is positively impacting the early stages of drug development by providing a straightforward way to genome engineering. This property also attracted attention for CRISPR application in the cell and gene therapy area.Expert opinion: CRISPR technology is rapidly becoming the preferred tool for genome engineering and nowadays it is hard to imagine the drug discovery pipeline without this technology. With the years to come, CRISPR technology will undoubtedly be further refined and will flourish into a mature technology that will play a key role in supporting genome engineering requirements in the drug discovery pipeline as well as in cell and gene therapy development.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.