Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Computational Analysis of Signal Peptide-Dependent Secreted Proteins in Saccharomyces cerevisiae

Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors

Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.

Genetic Modification of Baculovirus Expression Vectors

As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

Construction and Expression of Bivalent Membrane-anchored DNA Vaccine Encoding Sj14FABP and Sj26GST Genes

In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-terminal of human placental alkaline phosphatase （PLAP） downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.

Bioinformatic analysis and functional characterization of CFEM proteins in Setosphaeria turcica

Common in Fungal Extracellular Membrane(CFEM)domains are uniquely found in fungal extracellular membrane proteins which are important for pathogens.This study identified 13 StCFEM proteins in the genome of Setosphaeria turcica,the hemibiotrophic fungus that causes northern corn leaf blight.Sequence alignment and WebLogo analysis of their CFEM domains indicated that the amino acids were highly conserved and that,with the exception of StCFEM1,2,3,and 6,they contained eight cysteines.Phylogenic analysis suggested that these 13 proteins(StCFEM1–13)could be divided into 2 clades based on the presence of the trans-membrane domain.Six StCFEM proteins with a signal peptide and without a trans-membrane domain were considered as candidate effector proteins.The CFEM domain in the candidate effector proteins could form a helical-basket structure homologous to Csa2 in Candida albicans.Transcriptome analysis suggested that the 13 genes were expressed during fungal infection and a yeast secretion assay revealed that these candidate effectors were secreted proteins.It was also found that StCFEM3,4,and 5 couldn't affect BAX/INF1-induced programmed cell death(PCD)in Nicotiana benthamiana and while StCFEM12 could suppress INF1-induced PCD,it showed no effect on BAXinduced PCD.This study found that there were 13 members of the S.turcica CFEM protein family and that StCFEM12 was a candidate effector.This study laid the foundation for illustrating the roles of CFEM proteins during the pathogenic processes of phytopathogens.

Host-targeting-motif Harbored Secretary Proteins in Genome of Plant Pathogenic Fungus Botrytis cinerea

According to our previous study, saprophytic fungi Botrytis cinerea contained 579 predicted secretary proteins. Among them, we found that 122 of these proteins contained the highly conserved pathogenic-related host-targeting-motif RxLx within 100 residues adjacent to the signal peptide cleavage site. According to PEDNAT and COG of the GenBank database, the functions of this motif containing proteins included metabolism modification and cell secretion. We blasted them in GenBank and found 47.54% had highly conserved homologues in other species, among them 74.1% had putative functional domains. This suggests these proteins are presumably ancient and vertically transmitted within the species. Many of these domains belonged to proteins which played roles in the pathogenic process of other kinds of pathogens and some had already been proved to be pathogenic secretary proteins of Botrytis cinerea. So we postulated that proteins contained host-targeting-motif RxLx were candidates participating in the pathogenesis of Botrytis cinerea.

Identification and expression of the CEP gene family in apple (Malus×domestica)

Plant peptide hormones play important roles in plant growth and development. Among these hormones, the C-TERMINALLYENCODED PEPTIDE（CEP） belongs to a newly found peptide family that regulates root development in Arabidopsis as well as in other species. However, nothing is known about the CEP genes in apple（Malus×domestica, MdCEP）. In this study, a total of 27 apple CEP genes were identified through a genome-wide analysis and were phylogenetically divided into three classes（Ⅰ, Ⅱ and Ⅲ）. The predicted MdCEP genes were distributed across 10 of 17 chromosomes with different densities. Next, the gene structures and motif compositions of the MdCEP genes were analyzed. Subsequently, the expression analysis suggested that the MdCEP genes were highly activated in roots and that MdCEP23 may play an important role in regulating the growth and development of roots. Moreover, all of the MdCEP genes were responsive to multiple abiotic stresses, indicating that MdCEP genes may be involved with various aspects of physiological processes in apple. Nearly one-third of MdCEP genes had a significant response to low nitrogen treatment. Most of the MdCEP genes were up-regulated under stress, including mannitol, polyethylene glycol（PEG） and abscisic acid（ABA）, suggesting that MdCEP genes may be involved in the drought stress response. This study provides insight into the putative functions of the MdCEP genes using a genome-wide analysis of the CEP gene family.

COVID-19,liver dysfunction and pathophysiology:A conceptual discussion

The intra and extracellular pathways of hepatic injury by coronavirus disease 2019(COVID-19)are still being studied.Understanding them is important to treat this viral disease and other liver and biliary tract disorders.Thus,this paper aims to present three hypotheses about liver injury caused by COVID-19:(1)The interactions between severe acute respiratory syndrome coronavirus 2 spike protein and membrane receptors in the hepatocyte;(2)The dysbiosis and"gutliver axis"disruption in patients with serious clinical presentations of COVID-19;and(3)The inflammatory response exacerbated through the production of interleukins such as interleukin-6.However,despite these new perspectives,the pathophysiological process of liver injury caused by COVID-19 is still complex and multifactorial.Thus,understanding all these variables is a challenge to science but also the key to propose individualized and effective patient therapies.

CLE14 functions as a "brake signal" to suppress age-dependent and stress-induced leaf senescence by promoting JUB1-mediated ROS scavenging in Arabidopsis

Leaf senescence is an important developmental process in the plant life cycle and has a significant impact on agriculture.When facing harsh environmental conditions,monocarpic plants often initiate early leaf senes-cence as an adaptive mechanism to ensure a complete life cycle.Upon initiation,the senescence process is fine-tuned through the coordination of both positive and negative regulators.Here,we report that the small secreted peptide CLAVATA3/ESR-RELATED 14(CLE14)functions in the suppression of leaf senescence by regulating ROS homeostasis in Arabidopsis.Expression of the CLE14-encoding gene in leaves was signifi-cantly induced by age,high salinity,abscisic acid(ABA),salicylic acid,and jasmonic acid.CLE14 knockout plants displayed accelerated progression of both natural and salinity-induced leaf senescence,whereas increased CLE14 expression or treatments with synthetic CLE14 peptides delayed senescence.CLE14 pep-tide treatments also delayed ABA-induced senescence in detached leaves.Further analysis showed that over-expression of CLE14 led to reduced.ROS levels in leaves,where higher expression of ROS scavenging genes was detected.Moreover,CLE14 signaling resulted in transcriptional activation of JUB1,a NAC family tran-scription factor previously identified as a negative regulator of senescence.Notably,the delay of leaf senes-cence,reduction in H202 level,and activation of ROS scavenging genes by CLE14 peptides were dependent on JUB1.Collectively,these results suggest that the small peptide CLE14 serves as a novel"brake signal"to regulate age-dependent and stress-induced leaf senescence through JUB1-mediated ROS scavenging.

The CLE40 and CRN/CLV2 Signaling Pathways Antagonistically Control Root Meristem Growth in Arabidopsis

Differentiation processes in the primary root meristem are controlled by several signaling pathways that are regulated by phytohormones or by secreted peptides. Long-term maintenance of an active root meristem requires that the generation of new stem cells and the loss of these from the meristem due to differentiation are precisely coordinated. Via phenotypic and large-scale transcriptome analyses of mutants, we show that the signaling peptide CLE40 and the recep- tor proteins CLV2 and CRN act in two genetically separable pathways that antagonistically regulate cell differentiation in the proximal root meristem. CLE40 inhibits cell differentiation throughout the primary root meristem by controlling genes with roles in abscisic acid, auxin, and cytokinin signaling. CRN and CLV2 jointly control target genes that promote cell differentiation specifically in the transition zone of the proximal root meristem. While CRN and CLV2 are not acting in the CLE40 signaling pathway under normal growth conditions, both proteins are required when the levels of CLE40 or related CLE peptides increase. We show here that two antagonistically acting pathways controlling root meristem differentiation can be activated by the same peptide in a dosage-dependent manner.

PEP7 acts as a peptide ligand for the receptor kinase SIRK1 to regulate aquaporin-mediated water influx and lateral root growth

Plant receptors constitute a large protein family that regulates various aspects of development and responses to external cues.Functional characterization of this protein family and the identification of their ligands remain major challenges in plant biology.Previously,we identified plasma membrane-intrinsic sucrose-induced receptor kinase 1(SIRK1)and Qian Shou kinase 1(QSK1)as receptor/co-receptor pair involved in the regulation of aquaporins in response to osmotic conditions induced by sucrose.In this study,we identified a member of the elicitor peptide(PEP)family,namely PEP7,as the specific ligand of th receptor kinase SIRK1.PEP7 binds to the extracellular domain of SIRK1 with a binding constant of 1.44±0.79μM and is secreted to the apoplasm specifically in response to sucrose treatment.Stabilization of a signaling complex involving SIRK1,QSK1,and aquaporins as substrates is mediated by alterations in the external sucrose concentration or by PEP7 application.Moreover,the presence of PEP7 induces the phosphorylation of aquaporins in vivo and enhances water influx into protoplasts.Disturbed water influx,in turn,led to delayed lateral root development in the pep7 mutant.The loss-of-function mutant of SIRK1 is not responsive to external PEP7 treatment regarding kinase activity,aquaporin phosphorylation,water influx activity,and lateral root development.Taken together,our data indicate that the PEP7/SIRK1/QSK1 complex represents a crucial perception and response module that mediates sucrose-controlled water flux in plants and lateral root development.

Asymmetric cell division in plant development

Asymmetric cell division(ACD) is a fundamental process that generates new cell types during development in eukaryotic species.In plant development,post-embryonic organogenesis driven by ACD is universal and more important than in animals,in which organ pattern is preset during embryogenesis.Thus,plant development provides a powerful system to study molecular mechanisms underlying ACD.During the past decade,tremendous progress has been made in our understanding of the key components and mechanisms involved in this important process in plants.Here,we present an overview of how ACD is determined and regulated in multiple biological processes in plant development and compare their conservation and specificity among different model cell systems.We also summarize the molecular roles and mechanisms of the phytohormones in the regulation of plant ACD.Finally,we conclude with the overarching paradigms and principles that govern plant ACD and consider how new technologies can be exploited to fill the knowledge gaps and make new advances in the field.

Expression and significance of the vascular permeability factor in nasal polyps

目的探讨血管通透性因子(vascular permeability factor, VPF)在鼻息肉组织中的表达及意义.方法将9例鼻息肉标本及8例下鼻甲粘膜标本行VPF及其受体flk-1的免疫组化染色,光镜观查.结果 VPF在鼻息肉组织的血管内皮细胞和腺体细胞的表达明显高于下鼻甲组织(P<0.01,P<0.05),flk-1在血管内皮细胞的表达明显高于下鼻甲组织(P<0.01).结论 VPF对鼻息肉发生过程中组织极度水肿的产生可能有非常重要的作用.

PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein＇s function. We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups： chloroplast, thylakoid, mitochondrion, secretory pathway, and ＂other＂. When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL： http：//bioinformatics.biol.uoa.gr/PredSL/.

Structure-function relationship of the mammarenavirus envelope glycoprotein

Mammarenaviruses, including lethal pathogens such as Lassa virus and Junin virus, can cause severe hemorrhagic fever in humans. Entry is a key step for virus infection, which starts with binding of the envelope glycoprotein （GP） to receptors on target cells and subsequent fusion of the virus with target cell membranes. The GP precursor is synthesized as a polypeptide, and maturation occurs by two cleavage events, yielding a tripartite GP complex （GPC） formed by a stable signal peptide （SSP）, GP1 and GP2. The unique retained SSP interacts with GP2 and plays essential roles in virion maturation and infectivity. GP1 is responsible for binding to the cell receptor, and GP2 is a class I fusion protein. The native structure of the tripartite GPC is unknown. GPC is critical for the receptor binding, membrane fusion and neutralization antibody recognition. Elucidating the molecular mechanisms underlining the structure-function relationship of the three subunits is the key for understanding their function and can facilitate novel avenues for combating virus infections. This review summarizes the basic aspects and recent research of the structure-function relationship of the three subunits. We discuss the structural basis of the receptor-binding domain in GP1, the interaction between SSP and GP2 and its role in virion maturation and membrane fusion, as well as the mechanism by which glycosylation stabilizes the GPC structure and facilitates immune evasion. Understanding the molecular mechanisms involved in these aspects will contribute to the development of novel vaccines and treatment strategies against mammarenaviruses infection.

Transcriptome analyses of insect cells to facilitate baculovirus-insect expression

The High Five cell line （BTI-TN-5B1-4） isolated from the cabbage looper, Trichoplusia ni is an insect cell line widely used for baculovirus-mediated recombinant protein expression. Despite its widespread application in industry and academic laboratories, the genomic background of this cell line remains unclear. Here we sequenced the transcriptome of High Five cells and assembled 25,234 transcripts. Codon usage analysis showed that High Five cells have a robust codon usage capacity and therefore suit for expressing proteins of both eukaryotic- and prokaryotic-origin. Genes involved in glycosylation were profiled in our study, providing guidance for engineering glycosylated proteins in the insect cells. We also predicted signal peptides for transcripts with high expression abundance in both High Five and Sf21 cell lines, and these results have important implications for optimizing the expression level of some secretory and membrane proteins.