Recombinant Expression System Research Articles

Codon Optimization-based Whole-gene Scanning Identifies Hidden Nucleotides Essential for Bombyx mori Nucleopolyhedrovirus polyhedrin Hyperexpression

During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host’s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5ʹ proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5ʹ coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.

Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater.

The development of innovative methods for highly efficient production of recombinant proteins remains a prominent focus of research in the biotechnology field, primarily due to the fact that current commercial protein expression systems rely on expensive chemical inducers, such as isopropyl β-D-thiogalactoside (IPTG). In our study, we designed a novel approach for protein expression by creating a plasmid that responds to copper. This specialized plasmid was engineered through the fusion of a copper-sensing element with an optimized multiple cloning site (MCS) sequence. This MCS sequence can be easily customized by inserting the coding sequences of target recombinant proteins. Once the plasmid was generated, it was introduced into an engineered Escherichia coli strain lacking copA and cueO. With this modified E. coli strain, we demonstrated that the presence of copper ions can efficiently trigger the induction of recombinant protein expression, resulting in the production of active proteins. Most importantly, this expression system can directly utilize copper-containing industrial wastewater as an inducer for protein expression while simultaneously removing copper from the wastewater. Thus, this study provides a low-cost and eco-friendly strategy for the large-scale recombinant protein production. To the best of our knowledge, this is the first report on the induction of recombinant proteins using industrial wastewater.

Characterization of cleavage patterns and assembly of N-terminally modified GII.6 norovirus VP1 proteins.

When expressed in vitro, the major capsid protein VP1 of a norovirus (NoV) can self-assemble into virus-like particles (VLPs), and its N-terminus can tolerate foreign sequences without the assembly being affected. We explored the effects of adding an N-terminal sequence to the VP1 of a GII.6 NoV strain on its cleavage and assembly. Sequences of varying lengths derived from the minor capsid protein VP2 were added to the VP1 N-terminus. Using a recombinant baculovirus expression system, the fusion proteins were expressed, and their cleavage patterns and assembly were analyzed using mass spectrometry and transmission electron microscopy, respectively. All of the fusion proteins were successfully expressed and exhibited varying degrees of enzyme cleavage, most probably at the N-terminus. LC-MS results revealed that similar fragments were obtained for wild-type VP1 and fusion proteins, indicating that the cleavage sites were conserved. EM analysis indicated that VLPs of different sizes were successfully assembled for certain fusion proteins. The study data demonstrate that NoV VP1 can tolerate foreign sequences of a certain length at its N-terminus and that a conserved cleavage pattern exists, which might facilitate further investigation of the assembly and cleavage mechanisms of NoV.

Optimization of Expression and Purification of Recombinant One of Central Nervous System Enzyme (Acetylcholinesterase)

In general, the use of natural enzymes is very limited and they are not economical to use in industry. To solve this problem, the recombinant expression systems approach has been used. Optimization of production processes in heterologous expression systems is one of the most important activities in today's competitive industries. One of the key factors for the commercialization of recombinant proteins is the achievement of a low-cost culture medium available for the production of suitable cell biomass that can be used on an industrial scale. Commercialization of recombinant products requires process economics and cost reduction, and reducing the cost of the product is possible by using a cheaper culture medium, so decided optimization of expression and purification of recombinant acetylcholinesterase (EC.3.1.1.7), because it’s one of the body's vital enzymes, that by hydrolyzing acetylcholine, which is a neurotransmitter, terminates neural messages in the area of cholinergic synapses. Because the enzyme acetylcholinesterase produced from natural sources is not suitable for industrial purposes and semi-mass production due to low production efficiency and high costs of enzyme preparation and purification processes, optimizing the growth conditions of microorganisms capable of the production and secretion of the enzyme acetylcholinesterase are very important, both in terms of yield and economic costs. The present study seeks to increase productivity and reduce the cost of producing this enzyme by optimizing the growth medium and expression of pichia pastoris yeast cells that have genes related to this enzyme and also by optimizing the purification and mass production of this enzyme.

Functional expression and ligand identification of homo- and heteromeric Drosophila melanogaster CO2 receptors in the Xenopus laevis oocyte system.

Carbon dioxide (CO2) is an important olfactory cue in Drosophila melanogaster and can elicit both attractive and aversive behaviors. It is detected by gustatory receptors, Gr21a and Gr63a, found in the ab1C neuron in basiconic sensilla on the third antennal segment. Volatile substances that modulate the receptors' function are of interest for pest control. While several substances block ab1C neurons or mimic the activating effect of carbon dioxide, it is not known if these substances are indeed ligands of the CO2 receptor or might act on other proteins in the receptor neuron. In this study, we used the recombinant Xenopus laevis expression system and two-electrode voltage-clamp technology to investigate the receptor function. We found that application of sodium bicarbonate evokes large inward currents in oocytes co-expressing Gr21a and Gr63a. The receptors most likely form hetromultimeric complexes. Homomultimeric receptors of Gr21a or Gr63a are sufficient for receptor functionality, although oocytes gave significantly lower current responses compared to the probable heteromultimeric receptor. We screened for putative blockers of the sodium bicarbonate response and confirmed that some of the substances identified by spike recordings of olfactory receptor neurons, such as 1-hexanol, are also blockers in the Xenopus oocyte system. We also identified a new blocking substance, citronellol, which is related to insect repellents. Many substances that activate receptor neurons were inactive in the Xenopus oocyte system, indicating that they may not be ligands for the receptor, but may act on other proteins. However, methyl pyruvate and n-hexylamine were found to be activators of the recombinant Gr21a/Gr63a receptor.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

T‐PEP Related Presentation: Alzheimer’s disease pathogenic protein SORLA forms homodimer potentially required for its function

AbstractBackgroundSo far, many genetic and molecular studies have indicated that the single‐pass transmembrane receptor SORLA can cause Alzheimer’s disease (AD) when mutated, but the detailed mechanism of how SORLA is involved in the pathogenesis of AD is still unknown. SORLA has a unique domain architecture including a huge, multidomain extracellular region with a Vps10p domain and 6 tandem repeats of fibronectin‐type‐III (3Fn) domains plus a short cytoplasmic tail. Although this unique composition must be essential for its potential function as a guiding molecule for protein sorting inside cells, there has been limited information about its overall molecular architecture. We are focusing on the function of SORLA in guiding trafficking along with the retromer complex, and recently found SORLA seems to form homodimers on the cell surface, which correlates with its function.MethodWe employed a highly efficient recombinant expression system for extracellular proteins using mammalian cells that enables detailed biochemical analysis. Using this technique, we produced recombinant secreted protein samples of the whole ectodomain and some fragments derived from human SORLA, and investigated its dimerization property by pull‐down assay followed by SDS PAGE and native PAGE. To investigate the three‐dimensional architecture, we ran a structural prediction using the amino acid sequence of SORLA’s ectodomain only without its signal sequence, propeptide, transmembrane region, and cytoplasmic region, using the program AlphaFold2.ResultOur biochemical experiments showed that human SORLA has homodimer forming property through its Vps10p domain and 3Fn domains. For theVps10p domain especially, it is indicated that the major fraction forms a homodimer in solution. Based on current structural information, we successfully created a mutant that forms covalent bound homodimer with an artificially introduced intermolecular disulfide bond. Interestingly, the predicted model of the ectodomain shows a folded compact structure, indicating that SORLA’s ectodomain forms a large globular shape on the surface of cells.ConclusionOur biochemical experiments showed that SORLA has two putative, opposite‐facing dimeric interfaces. Along with the computational molecular prediction, we propose that SORLA forms polymeric fibril‐like high dimensional structure inside the tubular endosomal membrane, supported by the retromer oligomer from the cytoplasmic side.

Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3

Nicotinic acetylcholine (ACh) receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [215, 3, 159, 225, 259]. All nicotinic receptors are pentamers in which each of the five subunits contains 4 TM domains. Genes encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [120]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [159]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). Nicotinic ACh receptors contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the 4 TM domains [264, 87]; see also [106]). The high resolution crystal structure of the molluscan ACh binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g.[35]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94Â resolution [55], has revealed the orthosteric binding site in detail (reviewed in [215, 120, 39, 198]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states. Other nicotinic receptors are assembled as combinations of α(2-6) and β(2-4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [96, 93, 159]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [159]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [159] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [263]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [159]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [158, 9, 118]).The nicotinic receptor Subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally- and/or heterologously-expressed nACh receptor subtypes [143]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses- see also [46]).

Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research.

Generation of a DSF-Guided Refolded Bacterially Expressed Hemagglutinin Ectodomain of Influenza Virus A/Puerto Rico/8/1934 H1N1 as a Model for Influenza Vaccine Antigens.

Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some decades, efforts are being made to generate new efficient, flexible, and cost-effective technologies to be transferred into production. Our work describes the development of a model influenza hemagglutinin antigen that is capable of inducing protection against viral challenge in mice. High amounts of the H1 hemagglutinin ectodomain, HA18-528, were expressed in a bacterial system as insoluble inclusion bodies. Solubilization was followed by a thorough differential scanning fluorimetry (DSF)-guided optimization of refolding, which allows for fast and reliable screening of several refolding conditions, yielding tens of milligrams/L of folded protein. Structural and functional analysis revealed native-like folding as well as the presence of a mix of monomers and oligomers in solution. Mice immunized with HA18-528 were protected when exposed to influenza A virus as opposed to mice that received full-length denatured protein. Sera of mice immunized with HA18-528 showed both high titers of antigen-specific IgG1 and IgG2a isotypes as well as viral neutralization activity. These results prove the feasibility of the recombinant bacterial expression system coupled with DSF-guided refolding in providing influenza hemagglutinin for vaccine development.

Development of an Efficient Recombinant Protein Expression System in Clostridium saccharoperbutylacetonicum Based on the Bacteriophage T7 System.

Recombinant proteins have broad applications. However, there is a lack of a recombinant protein expression system specifically for large-scale production in anaerobic hosts. Here, we developed a powerful and stringently inducible protein expression system based on the bacteriophage T7 system in the strictly anaerobic solvent-producing Clostridium saccharoperbutylacetonicum. With the integration of a codon optimized T7 RNA polymerase into the chromosome, a single plasmid carrying a T7 promoter could efficiently drive high-level expression of the target gene in an orthogonal manner, which was tightly regulated by a lactose-inducible system. Furthermore, by deleting beta-galactosidase genes involved in lactose metabolism, the transcriptional strength was further improved. In the ultimately optimized strain TM-07, the transcriptional strength of the T7 promoter showed 9.5-fold increase compared to the endogenous strong promoter Pthl. The heterologous NADP+-dependent 3-hydroxybutyryl-CoA dehydrogenase (Hbd1) from C. kluyveri was expressed in TM-07, and the yield of the recombinant protein reached 30.4-42.4% of the total cellular protein, surpassing the strong protein expression systems in other Gram-positive bacteria. The relative activity of Hbd1 in the crude enzyme was 198.0 U/mg, which was 8.3-fold higher than the natural activity in C. kluyveri. The relative activity of the purified enzyme reached 467.4 U/mg. To the best of our knowledge, this study represents the first application of the T7 expression system in Clostridium species, and this optimized expression system holds great potential for large-scale endotoxin-free recombinant protein production under strictly anaerobic conditions. This development paves the way for significant advancements in biotechnology and opens up new avenues for industrial applications.

Drosophila X virus-like particles as delivery carriers for improved oral insecticidal efficacy of scorpion Androctonus australis peptide against the invasive fruit fly, Drosophila suzukii.

Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects, offering a promising insecticidal component for insect pest control. However, effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph, where they can act. Here, we investigated the potential of a novel nanocarrier, Drosophila X virus-like particle (DXV-VLP), for delivering a neurotoxin from the scorpion Androctonus australis Hector (AaIT) against the invasive pest fruit fly, Drosophila suzukii. Our results show that the fusion proteins of DXV polyproteins with AaIT peptide at their C-termini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system, and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions. In addition, the AaIT peptides displayed on DXV-VLPs retained their toxicity, as demonstrated in injection bioassays that resulted in severe mortality (72%) in adults after 72 h. When fed to adults, mild mortality was observed in the group treated with DXV-AaIT (38%), while no mortality occurred in the group treated with AaIT peptide, thus indicating the significant role of DXV-VLPs in delivering AaIT peptides. Overall, this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control. Moreover, it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.

Development and characterization of a plant-derived norovirus-like particle vaccine

BackgroundNorovirus (NoV) is the most common cause of diarrheal episodes globally. Issues with in vitro cultivation systems, genetic variation, and animal models have hindered vaccine development. Plant-derived virus-like particles (VLPs) may address some of these concerns because they are highly immunogenic, can be administered by different routes, and can be rapidly produced to accommodate emerging viral strains. MethodsNoV VLPs (NoVLP) composed of the surface viral protein (VP) 1 of the GI and GII genogroups were produced in Nicotiana benthamiana using an Agrobacterium tumefaciens-based recombinant transient expression system. Leaves from infiltrated plants were harvested and NoVLPs were extracted and purified. The safety and immunogenicity of the GII.4 NoVLP, the genotype currently causing most human disease, were subsequently examined in rabbits and mice. ResultsFifteen GI and GII NoVLPs were successfully expressed in N. benthamiana and were structurally similar to NoV virions, as determined by cryogenic transmission electron microscopy. The NoVLP was well-tolerated, with no local or systemic signs of toxicity in rabbits. Three intramuscular doses of the GII.4 NoVLP adjuvanted with aluminum hydroxide induced robust IgG titers, IgG-secreting cells, histo-blood group antigen blocking titers, and IFNγ-secreting T cells in mice. In addition to circulating antibodies, oral administration of the NoVLP in mice induced significant IgA levels in feces, indicative of a mucosal response. ConclusionsThe plant-made NoVLP vaccine was safe and immunogenic in mice and rabbits. Multi-modal vaccination, combining oral and intramuscular administration could be considered for future clinical development to maximize systemic and mucosal immune responses.

Production of HPV16-L1 Through BL21/pET32a Expression System

Background: The human papillomavirus (HPV) main capsid protein L1 is naturally capable of self-assembly as virus-like particles (VLPs). There are different recombinant protein expression systems, such as bacteria, yeast, insect, plant, and mammalian cells, for the generation of VLP-based candidate vaccines targeting various pathogens. In this study, we produced the HPV16-L1 protein by BL21/pET32a expression system, and VLP production was confirmed. Methods: The recombinant plasmid pET32/L1 was transformed into Escherichia coli BL21 and selected with ampicillin. The positive clones containing the recombinant plasmid pET32/L1 were assessed by restriction endonucleases HindIII and XhoI and nested polymerase chain reaction (PCR). The expression of HPV16-L1 fusion protein in Escherichia coli BL21 was identified by SDS-PAGE and western blotting. Electron microscopy was used to evaluate VLP formation. Results: A codon-optimized L1 gene was expressed in BL21 under the control of the T7/lac promoter. Purification of L1 protein was achieved after Ni NTA chromatography. The 60 kDa protein was detected in the lysates of BL21, recognized as HPV16- L1 protein by western blotting. The VLPs were confirmed using electron microscopy. Conclusion: In this study, we established an efficient recombinant E. coli expression system for the production of HPV 16- L1 protein. The generated L1 protein was correctly self-assembled into VLPs. Therefore, BL21/pET32a as a prokaryotic expression system is a potent tool for HPV16-L1 VLP production.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Engineering alkaline-stable barley stripe mosaic virus-like particles for efficient surface modification

Viruses and virus-like particles are powerful templates for materials synthesis because of their capacity for precise protein engineering and diverse surface functionalization. We recently developed a recombinant bacterial expression system for the production of barley stripe mosaic virus-like particles (BSMV VLPs). However, the applicability of this biotemplate was limited by low stability in alkaline conditions and a lack of chemical handles for ligand attachment. Here, we identify and validate novel residues in the BSMV Caspar carboxylate clusters that mediate virion disassembly through repulsive interactions at high pH. Point mutations of these residues to create attractive interactions that increase rod length ∼2 fold, with an average rod length of 91 nm under alkaline conditions. To enable diverse chemical surface functionalization, we also introduce reactive lysine residues at the C-terminus of BSMV coat protein, which are presented on the VLP surface. Chemical conjugation reactions with this lysine proceed more quickly under alkaline conditions. Thus, our alkaline-stable VLP mutants are more suitable for rapid surface functionalization of long nanorods. This work validates novel residues involved in BSMV VLP assembly and demonstrates the feasibility of chemical functionalization of BSMV VLPs for the first time, enabling novel biomedical and chemical applications.

Clonidine stimulates force of contraction via histamine H2 receptors in the human atrium

Clonidine has various clinical effects mediated by agonism of α1- or α2-adrenoceptors and the blocking of hyperpolarization-activated-nucleotide-gated pacemaker channels (HCN). It is unknown whether clonidine can also stimulate human cardiac histamine H2 receptors (hH2Rs). We used isolated electrically stimulated left and spontaneously beating right atrial preparations from mice overexpressing the hH2R specifically in the heart (H2-TG), and spontaneously beating right atrial preparations of guinea pigs for comparison. Moreover, we studied isolated electrically stimulated muscle strips from the human right atrium. Clonidine (1, 3, and 10 µM) increased force of contraction in isolated left atrial preparations from H2-TG mice. In contrast, clonidine reduced the spontaneous beating rate in right atrial preparations from H2-TG. Clonidine raised the beating rate in guinea pig right atrial preparations. Clonidine failed to increase the force of contraction but reduced beating rate in wild-type litter mate mice (WT). In WT, histamine failed to increase the force of contraction in left atrial preparations and beating rate in right atrial preparations. Clonidine (10 µM) increased the force of contraction in isolated human right atrial preparations. The positive inotropic effect in the human atrium was attenuated by cimetidine (10 µM). Clonidine increased the beating rate of the isolated spontaneously beating guinea pig right atrium and acted as a H2R partial agonist. Furthermore, clonidine showed binding to the guinea pig H2R (100 µM) using HEK cells in a recombinant expression system (pKi < 4.5) but hardly to the human H2R. These data suggest that clonidine can functionally activate cardiac human H2R.

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation.We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilized virus-like particles (VLPs) in Pichia pastoris.The stabilized VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilization, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralize virus in vitro. Therefore, anti-EVA71 neutralizing antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

Large-scale and cost-effective production of recombinant human serum albumin (rHSA) in transgenic Bombyx mori cocoons

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand.