Rapid human genomic DNA cloning into mouse artificial chromosome via direct chromosome transfer from human iPSC and CRISPR/Cas9-mediated translocation.

A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

A Modular Cloning Toolkit Including CRISPRi for the Engineering of the Human Fungal Pathogen and Biotechnology Host Candida glabrata.

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae-based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK (C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

The Ubiquitin E3 Ligase Nedd4 Regulates the Expression and Amyloid-ß Peptide Export Activity of P-Glycoprotein.

The ATP-binding cassette transporter, P-glycoprotein (P-gp), has been demonstrated to facilitate the clearance of amyloid-beta (Aß) peptides, exporting the neurotoxic entity out of neurons and out of the brain via the blood-brain barrier. However, its expression and function diminish with age and in Alzheimer's disease. P-gp is known to undergo ubiquitination, a post-translational modification that results in internalisation and/or degradation of the protein. NEDD4-1 is a ubiquitin E3 ligase that has previously been shown to ubiquitinate P-gp and reduce its cell surface expression. However, whether this effect translates into altered P-gp activity remains to be determined. siRNA was used to knockdown the expression of Nedd4 in CHO-APP cells. Western blot analysis confirmed that absence of Nedd4 was associated with increased P-gp protein expression. This was accompanied by increased transport activity, as shown by export of the P-gp substrate calcein-AM, as well as enhanced secretion of Aß peptides, as shown by ELISA. These results implicate Nedd4 in the regulation of P-gp, and highlight a potential approach for restoring or augmenting P-gp expression and function to facilitate Aß clearance from the brain.

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

Molecular cloning and functional characterization of KCNQ1 in shell biomineralisation of pearl oyster Pinctada fucata martensii.

KCNQ1, a voltage-gated potassium ion channel, plays an important role in various physiological processes, including osteoblast differentiation in higher animals. However, its function in lower invertebrates such as marine shellfish remains poorly understood. Pearl oysters, such as P. fucata martensii, are ideal for studying biomineralisation. In this study, a full-length cDNA of KCNQ1 from P. fucata martensii (PfKCNQ1) was obtained, and its function in shell formation was investigated. The full-length 3945 bp cDNA of PfKCNQ1 included an open reading frame (ORF) of 1944 bp encoding a polypeptide of 647 amino acids. Multiple sequence alignment revealed high homology with KCNQ1 from other species, with six transmembrane domains (S1 - S6) and a pore (P) region. Expression pattern analysis showed that PfKCNQ1 was expressed in all tested tissues, with highest expression in mantle and heart, and shell notching induced PfKCNQ1 expression. Silencing PfKCNQ1 expression inhibited PfKCNQ1 expression and downregulated four biomineralisation-related genes (Shematrin, Pif80, N16 and MSI60). Disordered crystals or "hollows" were visible in the shell ultrastructure by scanning electron microscopy following PfKCNQ1 knockdown. The results suggested that PfKCNQ1 may participate in or regulate biomineralisation and shell formation in pearl oyster.

Effect of pH on the structure and function of cyclin-dependent kinase 6.

Cyclin-dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0-4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0-11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0-8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.

Are there any good experiments that should not be done?

Is there any research that should not be done? Could you think of an experiment and then decide not to do it? These questions get to the heart of the power of modern genetics to mix up and alter genes.

Optimal Secretory Expression of Acetaldehyde Dehydrogenase from Issatchenkia terricola in Bacillus subtilis through a Combined Strategy.

Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 ∘C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Interaction of ONION2 ketoacyl CoA synthase with ketoacyl CoA reductase of rice.

BACKGROUND: Fatty acid elongases (FAEs), which catalyse elongation reactions of a carbon chain of very-long-chain fatty acids, play an important role in shoot development in rice. The elongation reactions consist of four sequential reactions catalysed by distinct enzymes, which are assumed to form an elongation complex. However, no interacting proteins of ONION1 (ONI1) and ONI2, which are ketoacyl CoA synthase catalyzing the first step and are required for shoot development in rice, are reported. METHODS AND RESULTS: In this study ketoacyl CoA reductase (KCR) that interacts with ONI1 and ONI2 was searched. A database search identified 10 KCR genes in the rice genome. Among the genes, the expression pattern of KCR1 was similar to that of ONI2. Yeast two-hybrid analysis showed interaction of ONI2 with KCR1, which was confirmed by GST pull-down assay. No interacting partner of ONI1 was identified. CONCLUSIONS: Our results suggest that ONI2 and KCR1 form an FAE complex that may play a role in biosynthesizing VLCFAs during shoot development.

Cloning and functional identification of setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1).

Plant somatic embryogenesis receptor-like kinases (SERK), members of leucine-rich repeat receptor-like kinases (LRR-RLKs) subfamily, are widely involved in plant growth, development and innate immunity. In this study, the setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1) was cloned by gateway technology, and transferred into a brasssinosteroid (BR) receptor mutant of Arabidopsis thaliana WS2 (bri1-5). After BL treatment, the transgenic plants could partially restore the phenotype of bri1-5. After Pst DC3000 treatment, the CFU value of SiSERK1 overexpression plant pathogen was between WS2 and bri1-5. Stomatal opening and plant height were also between them. Therefore, it is speculated that SiSERK1 gene is involved in BR signaling pathway and can improve the resistance of bri1-5 to Pst DC3000 through SA and NHP mediated systemic acquired resistance (SAR).

Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition.

Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01 Šand with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel ß-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.

Molecular cloning, heterologous expression, and in silico sequence analysis of Enterobacter GH19 class I chitinase (chiRAM gene).

BACKGROUND: Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli. METHODS AND RESULTS: The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met1-Ala23), a catalytic domain (Val83-Glu347 and the catalytic triad Glu149, Glu171, and Ser218), a proline-rich hinge region (Pro414 -Pro450), a polycystic kidney disease protein motif (Gly 465-Ser 533), a C-terminus chitin-binding domain (Ala553- Glu593), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1). CONCLUSIONS: Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization.

Analysis of opsin gene family of Crimson snapper (Lutjanus erythropterus).

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.

Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding ß-D-Galactose, N-Acetyl-D-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata).

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843 bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5 mM IPTG after 4 h incubation at 37 °C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35 kDa m.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26 mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of ß-pleated sheets and ß-turns in RbL protein structure. ß-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system.

Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU.

Cloning, expression and function analysis of trehalose-6-phosphate synthase gene from Marsupenaeus japonicu.

Trehalose is an important disaccharide that plays an important role in extreme environmental conditions. Trehalose-6-phosphate synthase (TPS) gene is the key gene for trehalose synthesis in Marsupenaeus japonicus. In this study, a TPS gene was isolated and characterized from M. japonicus. The full-length cDNA of TPS gene of M. japonicus (MjTPS) was 3308 bp, encoding 844 amino acids. The protein of the deduced MjTPS contained a glycol_transf_20 domain and a trehalose_PPase domain. The mRNA expression level of MjTPS was the highest in hepatopancreas. The further analysis found that MjTPS gene expression was up-regulated in a short time under low-salinity and high-nitrite stress, indicating that MjTPS gene had certain resistance to low-salinity and high-nitrite stress. Compared with the control group, both the expression of MjTPS and the trehalose content significantly decreased from 3 h to 24 h after MjTPS gene interference,. After RNAi, the mortality of M. japonicus increased, the expression level of MjTPS and the synthesis of downstream products decreased under low-salinity and high-nitrite stress, and what's more, the expression of immune genes PMO25, ERP, CD, HSP90, HSP70, HSP60, HMC and CLEC2 were significantly changed, implying that MjTPS might be participated in the immune response of M. japonicus. In addition, MjTPS gene silencing could affect the expression of CHI1 and CHS, suggesting that MjTPS might be involved in molting behavior of M. japonicus. These results provide new information for further studying the function of trehalose-6-phosphate synthase in shrimp.

Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.