Expression and Purification of Enterovirus Type 71 Polyprotein P1 using Pichia pastoris system

Enterovirus type 71(EV71) causes severe hand-foot-and-mouth disease(HFMD) resulting in hundreds of deaths of children every year;However,currently,there is no effective treatment for EV71.In this study,the EV71 poly-protein(EV71-P1 protein) gene was processed and cloned into the eukaryotic expression vector pPIC9k and then expressed in Pichia pastoris strain GS115.The EV71 P1 protein with a molecular weight of 100 kD was produced and secreted into the medium.The soluble EV71 P1 protein was purified by column chromatography with a recovery efficiency of 70%.The result of the immunological analysis showed that the EV71 P1 protein had excellent immunogenicity and could stimulate the production of EV71-VP1 IgG antibody in injected rabbits.We suggest that EV71-P1 protein is an ideal candidate for an EV71 vaccine to prevent EV71 infection.

茯苓多糖合成途径磷酸葡萄糖变位酶和UDP-葡萄糖焦磷酸化酶基因鉴定

采用已报道的灵芝磷酸葡萄糖变位酶(PGM)和UDP-葡萄糖焦磷酸化酶(UGPP)基因序列对茯苓基因组进行搜索比对,获得茯苓WcPGM和WcUGPP候选基因序列;以茯苓的cDNA为模板,成功克隆获得WcPGM和WcUGPP基因;随后利用pPIC9K构建2个基因的表达载体,并转化至毕赤酵母进行异源表达。序列分析表明WcPGM和WcUGPP基因全长分别为2021 bp和2144 bp,其中WcPGM基因含有5个外显子和4个内含子,编码564个氨基酸;WcUGPP基因含有11个外显子和10个内含子,编码502个氨基酸。氨基酸序列比对表明,WcPGM和WcUGPP与来源于绣球菌的ScPGM和ScUGPP的同源性分别为87%和91%。酶活测定表明,重组酶WcPGM可转化葡萄糖-1-磷酸(G1P)为葡萄糖-6-磷酸(G6P),酶活达1540 U·mL^(-1);重组酶WcUGPP可催化G1P和尿苷三磷酸(UTP)合成UDP-葡萄糖(UDP-Glc),酶活达660 U·mL^(-1),表明从茯苓中筛选到的2个酶WcPGM和WcUGPP具有PGM和UGPP活性。分子模拟表明,WcPGM在催化可逆反应过程中S114为磷酸供体和受体,R24和K379作为催化酸和碱实现底物的磷酸化和去磷酸化,而E366和S368可实现底物的2种朝向,保障了中间产物葡萄糖-1,6-二磷酸的翻转,确保了WcPGM的可逆反应;而WcUGPP活性中心的核苷酸结合Loop和糖结合Loop保障了底物的正确朝向,K390作为催化碱实现了底物UTP/G1P与UDP-Glc的可逆反应。该研究为茯苓多糖合成途径的解析和代谢工程提升茯苓多糖产量奠定了基础。

Expression and Characterization of a Thermostable Xylanase Gene xynA from a Themophilic Fungus in Pichia pastoris

The gene of xylanase （xynA） was amplified by RT-PCR from the total RNA of a themophilic fungus Thermomyces lanuginosus SY2. The sequence analysis showed that gene coding region of mature peptide contained 0.585 kb, which coded 194 amino acids. The putative amino acid sequence and DNA sequence of xylanase from T. lanuginosus SY2 （GenBank no.： GU166389） were 98.97 and 99.49% identical to the other T. lanuginosus （GenBank no.： U35436）. A recombinant plasmid pPIC9K-xynA was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Linearized pPIC9K-xynA was transformed into P. pastoris GS115 with the method of electroporation. The recombinant strain was identified by G418 selection and confirmed by PCR analysis. It was induced by 1.0% methanol at 28°C to express the recombinant xylanase. The results showed that the recombinant xylanase was secreted into extracellular fermentation liquid. The highest enzyme activity of 113.5 IU mL-1 and protein content of 889.7 μg mL-1 were detected for 216 h of induction. The optimal pH value and temperature of the enzyme activity was 5.5 and 65°C, respectively. The xylanase activity retained above 80% from pH value 2.5 to 8.5 for 48 h. The enzyme activity was above 85% at incubation temperature of 55°C.

High-level expression and purification of Plutella xylostella acetylcholinesterase in Pichia pastoris and its potential application

The acetylcholinesterase 2（AChE2）cloned from Plutella xylostella was first successfully expressed in methylotrophic yeast Pichia pastoris GS115.One transformant with high-level expression of the recombinant AChE（rAChE,23.2 U mL-1in supernatant）was selected by plating on increasing concentrations of antibiotic G418 and by using a simple and specific chromogenic reaction with indoxyl acetate as a substrate.The maximum production of r ACh E reached about 11.8 mg of the enzyme protein per liter of culture.The r ACh E was first precipitated with ammonium sulfate（50%saturation）and then purified with procainamide affinity column chromatography.The enzyme was purified 12.1-fold with a yield of 22.8%and a high specific activity of 448.3 U mg-1.It was sensitive to inhibition by methamidophos and pirimicarb,the calculated 50% inhibitory concentration（IC50）values of the two pesticides were 0.357 and 0.888 mg L-1,respectively,and the calculated 70% inhibitory concentration（IC70）values were 0.521 and 0.839 mg L-1,respectively.The results suggested that it has a potential application in the detection of pesticide residues.

Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products

The antifungal peptides, drosomycin （Drs） and its isoform drosomycin-like C （Drs-lC） from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains.

Expression of Recombinant Human Lysozyme-tachyplesin I(hLYZ-TP I)in Pichia Pastoris and Analysis of Antibacterial Activity

Antimicrobial peptides （AMPs） are making headlines in science because they demonstrate superior microbicidal characteristics compared to synthetic and semi-synthetic antibiotics.

High-yield expression of recombinant mouse coagulation factor Ⅶ in methylotrophic yeast Pichia pastoris

Objective: To explore high-yield secretory expression of recombinant mouse coagulation factor Ⅶ (rmF Ⅶ ) protein in Pichia pastoris (P. pastoris). Methods: The fragment of mF Ⅶ cDNA was amplified by PCR from a pcDNA3-mFⅦ plasmid. Then the cDNA fragment was subcloned into α-factor secretion signal open reading frame of pPIC9K secretory expression vector. The mutagenesis of mF Ⅶ was performed by Site-Direct Mutation and then verified by DNA sequencing. The yeast expression vector of rmF Ⅶ, named as pPIC9K-rmFⅦ, was linearized with Sac I and transferred into GS115 strains(his-Mut+)by electroporation. The recombinants were identified by direct PCR and selection on MM and MD plates. rmF Ⅶ was expressed in recombinant strains (his+Mut+) for 4 d. The expression level and activation of rmF Ⅶ in the BMMY medium were detected by SDS-PAGE and Western blot respectively. Results:pPIC9K-rmFⅦ was constructed and transferred to GS115 strains successfully. 48-hour post induction by methanol rmFⅦ protein was secreted into the culture supernatant. The molecular weight of the expressed products was shown to be about 46 kD by SDS-PAGE analysis. Western blot showed that the expressed rmF Ⅶ exhibited specificity and antigenicity. Conclusion: Since mFⅦ is considered as a tumor-targeting molecule , this study may provide a basis for further anti-tumor strategy on rmFⅦ.

Codon Optimization of SMAP-29 Gene and Its Expression in Pichia pastoris

[ Objective] This study aimed to optimize the cedon of antimicrobial peptide SMAP-29 gene and express SMAP-29 in Pichia pastoris. [ Method] Ac- cording to the published amino acid sequence of SMAP-29, a gene encoding SMAP-29 （sheep myeloid antibacterial peptides-29） mature peptide was synthesized and was biased codon usage of Pichia pastoris. The synthesized gene was inserted into Pichia pastoris expression vector pPIC3.5K, transformed into Pichia pastoris strain GSll5 by electmporation, and screened with G418 for high-copy recombinants. The methanol utility plus phenotype （Mut ＋ ） was selected with MM, MD and PCR. The correctly constructed recombinant strain was induced with methanol. Expression of SMAP-29 was analyzed by lysing the induced yeast cells with Tricine- SDS-PAGE. [ Result] After induced with methanol for 2 d, a 3.2 kD induced band was detected in the cell lysate which was consistent to the predicted molecular weight of SMAP-29 ; the expression products purified with gel filtration chromatography showed significant antibacterial effect against Staphyloccocus aureus and Mo- nilia albican but no significant antibacterial effect against Escherichia coli. [ Conclusion] This study laid the foundation for the application of SMAP-29 in biomedi- cine, agriculture and other fields.

Secretory expression and characterization of a recombinant-deleted variant of human hepatocyte growth factor in Pichia pastoris

AIM: To study the secretory expression of human hepatocyte growth factor (hdHGF) gene in Pichia pastoris.METHODS: The full-length gene of human cDNA encoding the deleted variant of hdHGF was cloned by RT-PCR and overlapping-fragment PCR technique using mRNA of human placenta as a template. The cloned hdHGF cDNA was inserted into the Escherichia coliyeast shuttle vector of pPIC9. The constructed plasmid,pPIC9-hdHGF, was transformed into the GS115 cells of the methylotrophic yeast, P pastoris, using a chemical method. The Mut+ transformants were screened to obtain high-expression strains by the test and analysis of expressed products of shake-flask culture. A secretory form of rhdHGF was made with the aid of the leader peptide sequence of Saccharomyces cerevisiae α-factor.RESULTS: The expressed products, which showed a band of molecular mass of about 80 ku, were observed on 15% SDS-PAGE and identified by Western blotting and N-terminal amino acid sequencing. In the high cell density culture of 5 L fermentor by fed-batch culture protocol, the cell biomass was reached at approximately 135 g (DCW)/L. The productivity of secreted total supernant protein concentration attained a high-level expression of more than 8.0 g/L and the ratio of rhdHGF band area was about 12.3% of the total band area scanned by SDS-PAGE analysis, which estimated that the product of rhdHGF was 500-900 mg/L.CONCLUSION: The P pastoris system represents an attractive tool of generating large quantities of hdHGF for both research and industrial purposes.

Antitumor activity of miR-188-3p in gastric cancer is achieved by targeting CBL expression and inactivating the AKT/mTOR signaling

BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.

Constitutive and Secretory Expression of the AiiA in Pichia pastoris Inhibits Amorphophallus konjac Soft Rot Disease

Amorphophallus konjac is an important economic crop widely cultivated in Southeast Asia and Africa. However, A. konjac is seriously infected by soft rot pathogen. The endocellular acyl homoserine lactonase (AiiA) which is generated by Bacillus species has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain recombinant yeast which produces AiiA protein. The recombinant yeast Pichia pastoris GS115 was constructed to constitutive expression of the AiiA gene. The results of reverse transcript PCR analysis showed that the AiiA gene was expressed successfully in the yeast. Proteins extracted from YPDS showed the highest inhibition efficacy to E. carotovora compared with the other two mediums (YPD and LB) under tested conditions.

Heterologous Expression of Mycobacterium tuberculosis Ag85B in Pichia pastoris

The DNA fragment encoding mature Mycobacterium tuberculosis major secretory protein Ag85B was inserted into the Pichia pastoris secretory expression vector pHBM905A, under the control of the AOX1 promoter. The recombinant plasmid pHBM905A-85B linearized by Sal I was introduced into Pichia pastoris strain GS115 by PEG1000 transformation method. After phenotype screening and PCR identification, the resulting GS115-pHBM905A-85B strain was cultivated and induced with methanol. The recombinant Ag85B protein in secreted form was attained with molecular weight of 35 × 10^3 approximately detected by SDS-PAGE and Western blot. EI,ISA experiment proved that the protein had good antigen specificity. Secretory expression of recombinant M. tuberculosis Ag85B in P. pastoris will open a door to mass production of the protein in heterologous host and allow ready evaluation of its immunological function.

Expression of ChIFN-α in Pichia pastoris and Optimization of Its Expression Condition

Because yeast codon preferred to synthesize the gene sequence of ChIFN-α mature peptide, Pichia pastoris expression system was connected into vector pPICgK, to construct recombinant plasmid pPIC9K-α. After digestion and linearization, the recombinant vector was cloned into P. pastor/s GS115, high copy transformant and its methanol utilization type were screened, and its expression conditions were optimized. The results showed that ChlFN-α successfully expressed in P. pastor/s, the expression amount of recombinant protein was the largest after induced expression in 0.5 % methanol containing 2% acid hydrolyzed casein at 28 ℃ for 72 h, and its antiviral activity was the highest.

Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris

The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.

Fusion Expression of Glucoamylase and Xylanase in Pichia pastoris

Employing RT-PCR amplification method, the mature pepfide coding sequence of glucoamylase （amyA） gene was amplified from total RNA of Talaro- myces emersonii and the xylanase （xyrut） gene from genomic DNA ofPaenibacillus sp. H10-3. The result showed that the mature peptide sequence of amyA is 1857 bp long and encodes 618 amino acids; and the mature peptide sequence of xynA is 636 bp long and encodes 211 amino acids. Then these two genes were spliced by overlap extension PCR （ SOE-PCR）, yielding fusion gene amyA-l-xynA. The fusion gene amyA-l-xynA was then cloned into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. The recombinant plasmid pPICg-amyA-l-xynA was linearized and transformed into Pichia pastoris GSll5 via electric shock, yielding engineering strain ALX2. The maximum yields of glucoamylase and xylanase in ALX2 fermentation supernatant were determined as 10.7 and 51.8 U/ml, respectively.

Exercise-induced modulation of miR-149-5p and MMP9 in LPS-triggered diabetic myoblast ER stress: licorice glycoside E as a potential therapeutic target

Background:This study explores the relationship between endoplasmic reticulum(ER)stress and diabetes,particularly focusing on the impact of physical exercise on ER stress mechanisms and identifying potential therapeutic drugs and targets for diabetes-related sepsis.The research also incorporates traditional physical therapy perspectives,emphasizing the genomic insights gained from exercise therapy in disease management and prevention.Methods:Gene analysis was conducted on the GSE168796 and GSE94717 datasets to identify ER stress-related genes.Gene interactions and immune cell correlations were mapped using GeneCard and STRING databases.A screening of 2,456 compounds from the TCMSP database was performed to identify potential therapeutic agents,with a focus on their docking potential.Techniques such as luciferase reporter gene assay and RNA interference were used to examine the interactions between microRNA-149-5p and MMP9.Results:The study identified 2,006 differentially expressed genes and 616 miRNAs.Key genes like MMP9,TNF-α,and IL1B were linked to an immunosuppressive state.Licorice glycoside E demonstrated high affinity for MMP9,suggesting its potential effectiveness in treating diabetes.The constructed miRNA network highlighted the regulatory roles of MMP9,IL1B,IFNG,and TNF-α.Experimental evidence confirmed the binding of microRNA-149-5p to MMP9,impacting apoptosis in diabetic cells.Conclusion:The findings highlight the regulatory role of microRNA-149-5p in managing MMP9,a crucial gene in diabetes pathophysiology.Licorice glycoside E emerges as a promising treatment option for diabetes,especially targeting MMP9 affected by ER stress.The study also underscores the significance of physical exercise in modulating ER stress pathways in diabetes management,bridging traditional physical therapy and modern scientific understanding.Our study has limitations.It focuses on the microRNA-149-5p-MMP9 network in sepsis,using cell-based methods without animal or clinical trials.Despite strong in vitro findings,in vivo studies are needed to confirm licorice glycoside E’s therapeutic potential and understand the microRNA-149-5p-MMP9 dynamics in real conditions.

Expression of human acidic fibroblast growth factor in Pichia pastoris

Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.

四联鲎抗菌肽Tachyplesin Ⅱ毕赤酵母重组质粒构建及其表达

利用重组毕赤酵母表达四联鲎抗菌肽Tachyplesin Ⅱ以实现高效制备。根据鲎抗菌肽Tachyplesin Ⅱ的氨基酸序列,以及胰弹性蛋白酶和羧肽酶A的特异性裂解位点,设计四联鲎抗菌肽(4Tp Ⅱ)序列,按照毕赤酵母密码子偏好性进行优化,经EcoR Ⅰ和Not Ⅰ双酶切后构建重组质粒pPICZαA-4Tp Ⅱ,电转至毕赤酵母X-33,利用Zeocin抗性筛选阳性转化子,经甲醇诱导表达后SDS-PAGE分析。成功构建重组质粒pPICZαA-4Tp Ⅱ,并筛选出阳性转化子,经30℃、250 r/min、1%甲醇诱导表达5 d后,获得重组蛋白,可为高效制备鲎抗菌肽Tachyplesin Ⅱ奠定基础。

Expression, Characterization and Antimicrobial Ability of a Variant T4 Lysozyme in Pichia pastoris

T4 lysozyme was engineered with disulfide bonds and expressed in Pichia pastoris. The secreted proteins were purified and made into powder by lyophilization. Recombinant protein purity was more than 70% measured by HPLC. The lytic activity of variant T4-lysozyme was measured by the lysis of the cell wall of Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, Ralstonia solanacearum comb.nov, Clavibacter michiganensis subsp. michiganensis, X. campestris pv. malvacearum, Fusarium oxysporium sp. vasinfectum, Verticillium dahliae kleb. Inhibition zone assay showed that variant T4 lysozyme significantly inhibited X. o. oryzicola and X. c. malvacearum. The antifungal activities of this protein against F. o. vasinfectum and V. d. kleb were also analyzed.