Recombinant Protein Expression Research Articles

Employing Engineered Enolase Promoter for Efficient Expression of Thermomyces lanuginosus Lipase in Yarrowia lipolytica via a Self-Excisable Vector.

Yarrowia lipolytica is progressively being employed as a workhouse for recombinant protein expression. Here, we expanded the molecular toolbox by engineering the enolase promoter (pENO) and developed a new self-excisable vector, and based on this, a combined strategy was employed to enhance the expression of Thermomyces lanuginosus lipase (TLL) in Y. lipolytica. The strength of 11 truncated enolase promoters of different length was first identified using eGFP as a reporter. Seven of the truncated promoters were selected to examine their ability for driving TLL expression. Then, a series of enolase promoters with higher activities were developed by upstream fusing of different copies of UAS1B, and the recombinant strain Po1f/hp16e100-tll harboring the optimal promoter hp16e100 obtained a TLL activity of 447 U/mL. Additionally, a new self-excisable vector was developed based on a Cre/loxP recombination system, which achieved efficient markerless integration in Y. lipolytica. Subsequently, strains harboring one to four copies of the tll gene were constructed using this tool, with the three-copy strain Po1f/3tll showing the highest activity of 579 U/mL. The activity of Po1f/3tll was then increased to 720 U/mL by optimizing the shaking flask fermentation parameters. Moreover, the folding-related proteins Hac1, Pdi, and Kar2 were employed to further enhance TLL expression, and the TLL activity of the optimal recombinant strain Po1f/3tll-hac1-pdi-kar2 reached 1197 U/mL. By using this combined strategy, TLL activity was enhanced by approximately 39.9-fold compared to the initial strain. Thus, the new vector and the combined strategy could be a useful tool to engineer Y. lipolytica for high-level expression of heterologous protein.

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins.

Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24°C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Feed Supplemented with Aronia melanocarpa (AM) Relieves the Oxidative Stress Caused by Ovulation in Peak Laying Hens and Increases the Content of Yolk Precursors.

The continuous ovulation of laying hens during the peak period is likely to cause oxidative stress, resulting in a reduction in the laying cycle over time. The aim of this study was to evaluate the antioxidant effects of Aronia melanocarpa (AM) in the diet and its effect on the yolk precursor content caused by ovulation in laying hens during the peak period. A total of 300 25-week-old Roman brown laying hens were randomly divided into five groups with six replicates in each group, 10 in each replicate. The control group was fed a basal diet, the positive control group was fed a Vitamin C (VC) plus basal diet, and the experimental group was fed 1%, 4%, and 7% doses of AM plus diet according to the principle of energy and nitrogen requirements, which lasted eight weeks. At the end of the study, the egg quality, biochemical, and antioxidant markers, as well as mRNA and protein expressions, were evaluated to determine the potential signaling pathways involved. Results showed that the addition of AM to the feed increased the weight of laying hens at the peak of egg production and improved egg quality. The biochemical markers, as well as the antioxidant parameters in the serum, liver, and ovarian tissues, were ameliorated. The gene and protein expression of recombinant kelch-like ECH-associated protein 1 (Keap1) in the liver and ovarian tissues was decreased, while nuclear factor erythroid-2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression was increased. The feed supplemented with AM also increased the estrogen contents and lipid parameters, as well as the gene and protein expressions related to the yolk precursor. Feed supplemented with AM could improve the egg quality and the oxidative stress caused by the ovulation process of laying hens during the peak egg production period by activating the Keap1/Nrf2 signaling pathway. These results suggest that the feed supplemented with 1% AM and 4% AM can improve egg production in peak laying hens.

Apilimod enhances specific productivity in recombinant CHO cells through cell cycle arrest and mediation of autophagy.

Chinese hamster ovary (CHO) cells are expected to acquire the ability to produce higher recombinant therapeutic protein levels using various strategies. Genetic engineering targeting the cell cycle and autophagy pathways in the regulation of cell death in CHO cell cultures has received attention for enhancing the production of therapeutic proteins. In this study, we examined the small-molecule compound apilimod, which was found to have a positive influence on recombinant protein expression in CHO cells. This was confirmed by selective blocking of the cell cycle at the G0/G1 phase. Apilimod treatment resulted in decreased expression of cyclin-dependent kinase 3 (CDK3) and Cyclin C and increased expression of cyclin-dependent kinase suppressor p27Kip1, which are critical regulators of G1 cell cycle progression and important targets controlling cell proliferation. Furthermore, total transcription factor EB (TFEB) was lower in apilimod-treated CHO cells than in control cells, resulting in decreased lysosome biogenesis and autophagy with apilimod treatment. These multiple effects demonstrate the potential of apilimod for development as a novel enhancer for the production of recombinant proteins in CHO cell engineering.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli

Expression of affinity-tagged recombinant proteins for crystallography, protein–protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10–50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

Optimising expression and extraction of recombinant proteins in plants.

Recombinant proteins are of paramount importance for research, industrial and medical use. Numerous expression chassis are available for recombinant protein production, and while bacterial and mammalian cell cultures are the most widely used, recent developments have positioned transgenic plant chassis as viable and often preferential options. Plant chassis are easily maintained at low cost, are hugely scalable, and capable of producing large quantities of protein bearing complex post-translational modification. Several protein targets, including antibodies and vaccines against human disease, have been successfully produced in plants, highlighting the significant potential of plant chassis. The aim of this review is to act as a guide to producing recombinant protein in plants, discussing recent progress in the field and summarising the factors that must be considered when utilising plants as recombinant protein expression systems, with a focus on optimising recombinant protein expression at the genetic level, and the subsequent extraction and purification of target proteins, which can lead to substantial improvements in protein stability, yield and purity.

Nuclear localization signal peptides enhance genetic transformation of Dunaliella salina.

Dunaliella salina (D. salina) expression system shows a very attractive application prospect, but it currently has a technical bottleneck, namely the low or unstable expression of recombinant proteins. Given the characteristics of cell-penetrating peptides or/and nuclear localization signal (NLS) peptides, this study is the first attempt to improve the transformation rate of foreign gene with trans-activating transcriptional (TAT) protein or/and NLS peptides. Using salt gradient method, exogenous plasmids were transferred into D. salina cells with TAT or TAT/NLS complexes simultaneously. The β-glucuronidase gene expression was identified by means of histochemical stain and RT-qPCR detection. Through observation with light microscope, TAT-mediating cells exhibit an apparent cytotoxicity even at ratios of 0.5, no significant toxicity was noted in the TAT/plasmid/NLS complex group. It is obvious that with the addition of peptides the toxicity decreases significantly. Histochemical staining showed that the transformants presented blue color under light microscope, but the negative control and blank control are not. Furthermore, based on a TAT/plasmids ratio of 4 with 10µg NLS peptides mediation, RT-qPCR results demonstrated that the transcripts of target gene were increased by 269 times than that of control group. This study demonstrated that combination of TAT and NLS peptides can significantly improve the transformation rate and expression level of foreign gene in D. salina system. It offers a promising way for promoting the application and development of D. salina bioreactor.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

Expression of Influenza M2e-NP Recombinant Fusion Protein in Escherichia coli BL21 (DE3) and Its Binding to Antibodies

The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The M2e gene was fused to the 5'-end of the NP gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the M2e-NP fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into Escherichia coli (E. coli) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.

Design, cloning and expression of a synthetic gene encoding a grouper iridovirus major capsid protein

Synthetic biology approaches can be adopted to successfully redesign, clone and express recalcitrant proteins from viruses in a bacterial expression system. The grouper iridovirus major capsid proteins (GI-MCP) is a surface coat protein which has the potential for application as an antigen for the development of vaccines which confer immunity against GI. The native amino acid coding region of the GI-MCP does not lend itself to expression in the Escherichia coli (BL21) (DE3) platform due to the presence of internal motifs which represent the start and stop codons as well as secondary structures. This manuscript reports on the redesigning, cloning and expression of a synthetic GI-MCP in E. coli. The native GI-MCP protein coding region was retrieved from the NCBI GenBank. The sequence was codon optimized, internal start and stop codons were modified and potential secondary structures were resolved. Both the native and modified GI-MCP coding DNA sequences were synthesized and ligated onto an expression plasmid, pET22b (+) followed by transformation, cloning and expression in E. coli (BL21) (DE3). Induction of protein expression was carried out at 12°C, 20°C and 37°C to assess protein solubility. The gene encoding the native GI-MCP failed to express under any of the culture conditions, however, the modified synthetic gene encoding the GI-MCP expressed the recombinant iridovirus coat protein (rGI-MCP) under all the conditions. Gene design and synthesis offer an ideal solution for the recombinant expression of viral proteins in bacterial expression systems for the industrial production of viral antigens.

Recombinant protein condensation inside E.coli enables the development of building blocks for bioinspired materials engineering - Biomimetic spider silk protein as a case study.

Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the mimetic spider silk mini-spidroin NT2RepCT in Escherichia coli. The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E.coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies.

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

Recombinant Expression of Human IL-33 Protein and Its Effect on Skin Wound Healing in Diabetic Mice.

Chronic refractory wounds are one of the complications of diabetes mellitus that require effective therapy. The dermal-wound-healing property of IL-33 in diabetics is little understood. Therefore, this study aimed to express recombinant humanized mature IL-33 (rhmatIL-33) in Escherichia coli BL21 (DE3) and demonstrate its efficacy on dermal wounds in streptozotocin (STZ)-induced diabetic and nondiabetic mice by the dorsal incisional skin wound model. Results revealed that the rhmatIL-33 accelerated the scratch-healing of keratinocytes and fibroblasts at the cellular level. The wounds of diabetic mice (DM) showed more severe ulceration and inflammation than wild-type mice (WT), and the exogenous administration of rhmatIL-33 increased wound healing in both diabetic and wild-type mice. Compared with the up-regulation of endogenous IL-33 mRNA after injury in WT mice, the IL-33 mRNA decreased after injury in DM mice. Exogenous IL-33 administration increased the endogenous IL-33 mRNA in the DM group but decreased the IL-33 mRNA expression level of the WT group, indicating that IL-33 plays a balancing role in wound healing. IL-33 administration also elevated ILC2 cells in the wounds of diabetic and non-diabetic mice and improve the transcript levels of YM1, a marker of M2 macrophages. In conclusion, Hyperglycemia in diabetic mice inhibited the expression of IL-33 in the dermal wound. Exogenous addition of recombinant IL-33 promoted wound healing in diabetic mice by effectively increasing the level of IL-33 in wound tissue, increasing ILC2 cells, and accelerating the transformation of macrophage M1 to M2 phenotype.

M6A-SAC-seq for quantitative whole transcriptome m6A profiling.

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m6A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233-129,263 sites using as little as 2 ng of poly A+ RNA. These identified sites correspond well with previous m6A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m6A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.

Generating a construct harboring the rpb1 promoter for enhancing recombinant protein expression in the medicinal mushroom Cordyceps militaris

Cordyceps militaris is a valuable medicinal mushroom that contains numerous bioactive ingredients such as cordycepin, adenosine, pentostatin, polysaccharides, and carotenoids. However, the contents of these substances in wild-type strains of C. militaris are relatively low. Recently, the genome of C. militaris has been fully sequenced, and genes involved in the biosynthesis of beneficial constituents have been identified. Therefore, research on enhancing the biosynthesis of the bioactive compounds in this fungus by recombinant DNA technology has advantages. In the present study, the authors have successfully generated a binary vector with a T-DNA region carrying a construct regulated by the native rpb1 promoter that can enhance gene expression in C. militaris. The T-DNA structure from the binary vector was then transferred into the C. militaris genome by Agrobacterium tumefaciens-mediated transformation. Analysis of some fungal transformants by PCR using the specific primer pairs indicated that the T-DNA structure was successfully integrated into the fungal genome. Observation of the transformants under fluorescence microscopy confirmed the overexpression of the DsRed fluorescent protein under the regulation of the rpb1promoter. The binary vector carrying the rpb1 promoter constructed in this study can be employed to promote the expression of target genes in the medicinal mushroom C. militaris.

Recombinant protein expression of Magnaporthe oryzae MGG06069 and its polysaccharide monooxygenase domain in Escherichia coli

MGG06069 is a polysaccharide monooxygenase protein that originated from the fungusthat causes rice blast, Magnaporthe oryzae. It has the potential to speed up thecellulolysis process, which can be beneficial for industry. In this study, the full-lengthMGG06069 protein and its polysaccharide monooxygenase domain were expressed inEscherichia coli in order to produce recombinant proteins on a large scale for furtherenzymatic experiment. It was shown that the amount of the recombinant polysaccharidemonooxygenase domain protein was much higher compared to that of the full-lengthprotein. In addition, only one band of expected size was obtained for the recombinantPMO domain protein. These results suggest the purified polysaccharidemonooxygenase domain of MGG06069 that can be used for enzymatic assaysin the future.® 2022 Journal of Science and Technology - NTTU

Plant-based expression platforms to produce high-value metabolites and proteins.

Plant-based heterologous expression systems can be leveraged to produce high-value therapeutics, industrially important proteins, metabolites, and bioproducts. The production can be scaled up, free from pathogen contamination, and offer post-translational modifications to synthesize complex proteins. With advancements in molecular techniques, transgenics, CRISPR/Cas9 system, plant cell, tissue, and organ culture, significant progress has been made to increase the expression of recombinant proteins and important metabolites in plants. Methods are also available to stabilize RNA transcripts, optimize protein translation, engineer proteins for their stability, and target proteins to subcellular locations best suited for their accumulation. This mini-review focuses on recent advancements to enhance the production of high-value metabolites and proteins necessary for therapeutic applications using plants as bio-factories.

Optimization of purification conditions and study of antigenic properties of recombinant nucleocapsid protein of different SARS-CoV-2 strains

BACKGROUND: In the context of the constant manifestation of new SARS-CoV-2 strains and the need to determine the immunogenicity of new variants of antiviral vaccines, it is necessary to create diagnostic test systems based on conservative viral proteins. The SARS-CoV-2 nucleocapsid protein can be considered as a candidate antigen. However, the relevance of existing test systems based on it for determining the titer of antibodies produced in response to infection by recently emerging strains is unknown.
 AIM: The goal is to optimize the conditions for obtaining recombinant N proteins of various SARS-CoV-2 strains and to analyze the possibility of creating ELISA test systems based on them.
 MATERIALS AND METHODS: Bacterial strains producing N proteins were obtained by amplifying the corresponding genes and ligating them into the pETDuet-1 expression vector. Expression was induced at 20 or 37C for 1, 2, 4, or 20 h using inducer (IPTG) concentrations of 0.1 mM or 0.5 mM with or without the addition of 3% ethanol. Proteins were purified from biomass by metal affinity chromatography and used as antigens for the detection of antiviral antibodies by ELISA.
 RESULTS: It was found that the concentration of the inductor sufficient for the expression of recombinant proteins is 0.1 mM, the induction time is 1 h, and the required temperature is 37 C. The influence of the presence of ethanol as an expression-stimulating reagent was not revealed. When determining the titers of antiviral antibodies using the obtained proteins, cross-reactivity of serums of COVID-19 convalescents was established regarding to antigens of various SARS-CoV-2 strains.
 CONCLUSIONS: The possibility of effective induction of protein synthesis at a minimum concentration of the inducer and cultivation time indicates the economy of its production, and antigen recognition by antiviral antibodies indicates a native structure. Cross-reactivity of the blood sera of convalescents indicates the slow character of the evolution of the antigenic properties of the SARS-CoV-2 N protein. Thus, the purified proteins can be used as a basis for development of diagnostic test systems.