Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

Phenotypical statin-associated immune-mediated necrotizing myositis with histological features of inclusion body myositis

Introduction:Statin-associated immune-mediated necrotizing myositis(IMNM)is a rare but distinct idiopathic inflammatory myopathy(IIM)that requires early recognition and intervention to prevent irreversible muscle damage.It is typically characterized by active statin use,elevated creatinine kinase(CK)levels,proximal muscle weakness,and at times,a positive 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase(HMGCR)antibody.Treatment includes immediate discontinuation of the statin and may include corticosteroids,intravenous immunoglobulin(IVIG),and/or immunosuppressive therapy.Inclusion body myositis(IBM),another distinct IIM,also presents with elevated CK levels but with insidious onset of distal upper and proximal lower extremity weakness and is typically refractory to treatment.Case Description:A 64-year-old female patient presented with proximal muscle weakness,elevated CK levels,and a positive HMGCR antibody in the setting of statin use with muscle pathology suggestive of both statinassociated IMNM and IBM.She responded to subcutaneous methotrexate and a slow prednisone taper over several months,however,will require close monitoring for symptoms associated with either disease.Conclusion:In conclusion,we report a case of muscle weakness with muscle pathology demonstrating both statin-associated IMNM and IBM.This case highlights the importance of understanding the clinical and pathological features of statin-associated IMNM and IBM.

Effect of Freeze-Thaw and Urea in Solubility of GPC3-Csub Protein Expressed in Escherichia coli

Glypican-3 is a protein encoded by the Glypican-3 gene located on human X chromosome (Xq26), composed of two subunits, a 40 kDa N-terminal subunit, and a 30 kDa C-terminal subunit. Glypican-3 is a currently potential target molecule for liver cancer treatments because of its over-expression and growth effects on hepatocellular carcinoma (HCC). This study examined the expression and purification of a C-terminal subunit of Glypican-3 protein (GPC3-Csub) due to its application in both diagnosis and therapy for hepatocellular carcinoma. The gene encoding for GPC3-Csub was successfully cloned into plasmid pET28a fused with an affinity tag composed of six consecutive histidine residues (His-tag). Recombinant protein GPC3-Csub was expressed in Escherichia coli BL21 (DE3) in the condition of adding 3% ethanol with IPTG induction. GPC3-Csub was extracted using repeated freeze-thaw cycles with lysozyme, and inclusion bodies were solubilized by 8M Urea, SDS 10% in pH 12. His-tag fused GPC3-Csub proteins allowed it to be purified by affinity chromatography method using the Nickel-nitrilotriacetic acid (Ni-NTA) column. High expression of GPC3-Csub was confirmed by Coomassie staining and western-blot. GPC3-Csub could be isolated with a Ni-NTA column and have a purity of about 90%.

Refolding and Purification of Yeast Acetyl-CoA Carboxylases CT Domain Expressed as Inclusion Bodies in Escherichia coli

Acetyl-CoA carboxylase（ACCase） is a crucial enzyme in fatty acid synthesis, by regulating the first committed step in the process. Therefore, it is a potential target for the development of new compounds against obesity or as herbicides. The cDNA encoding yeast ACCase CT domains（YCTs） from Saccharvmyces cerevisiae was amplified by RT-PCR and inserted into the vector PET28a（＋） for bacterial expression of YCT fused to N-terminal His-tag（YCT-his6）. YCTs-his6 was expressed in Escherichia coli BL21（DE3） PLys as inclusion bodies, which was solubilized in 8 mol/L urea. Ni-agarose chromatography was used to purify the inclusion bodies under denaturing condition. Correct refolding was achieved via systematic dialysis to remove the denaturant gently in the presence of 0.5 mmol/L Triton X-100. The low concentration Triton X-100 was included in the refolding buffer to increase the solubilization and enhance dimeric formation of refolding proteins. The activity of the refolded YCT-his6 was 1.2 U/rag as measured in a spectrophotometric assay using malonyl-CoA as the substrate. To our knowledge, it is the first time that the bioactive YCT-his6 has been expressed successfully in E. coli and isolated from their inclusion bodies.

Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

[Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

A New Protocol for Solubilization, Refolding and Purification of Recombinant Human Granulocyte Colony-stimulating Factor in Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.

PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment

Inclusion bodies(IBs)of respiratory syncytial virus(RSV)are formed by liquid-liquid phase separation(LLPS)and contain internal structures termed“IB-associated granules”(IBAGs),where anti-termination factor M2-1 and viral mRNAs are concentrated.However,the mechanism of IBAG formation and the physiological function of IBAGs are unclear.Here,we found that the internal structures of RSV IBs are actual M2-1-free viral messenger ribonucleoprotein(mRNP)condensates formed by secondary LLPS.Mechanistically,the RSV nucleoprotein(N)and M2-1 interact with and recruit PABP to IBs,promoting PABP to bind viral mRNAs transcribed in IBs by RNArecognition motif and drive secondary phase separation.Furthermore,PABP-eIF4G1 interaction regulates viral mRNP condensate composition,thereby recruiting specific translation initiation factors(eIF4G1,eIF4E,eIF4A,eIF4B and eIF4H)into the secondary condensed phase to activate viral mRNAs for ribosomal recruitment.Our study proposes a novel LLPS-regulated translation mechanism during viral infection and a novel antiviral strategy via targeting on secondary condensed phase.

Clinical Diagnosis Technique of Goat Pox Disease

[Objective] The aim was to provide theoretical basis for effective prevention of goat pox disease.[Method] 5 cases of infected goats were diagnosed for goat pox with microbiology examination.The poxes on their skin,rumen,reticulum,omasum,abomasum and submandibular lymph nodes,bronchial lymph nodes,lung and spleen were macroscopically and microscopically observed with pathanatomical and histopathological technique.[Result] Poxes on skin mainly showed ashen hemisphere state and gave prominence to the surface of skin; some cases had hemorrhage in the poxes and showed dark purplish red.Poxes on gastric mucosa showed ashen.Cytoplasmic inclusion body could be all observed in epithelial cells of the poxes and macrphages of lymph node,lung and spleen.[Conclusion] Poxes on skin,lung and the surface of gastric mucosa as well as cytoplasmic inclusion body in the epithelial cells of pox and the macrphages of lymphoid organs were the especial pathochanges of goat pox,which could be taken as the proof of goat pox＇s clinic diagnisis.

The role of sequestosome 1/p62 protein in amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are multifaceted diseases with genotypic,pathological and clinical overlap.One such overlap is the presence of SQSTM1/p62 mutations.While traditionally mutations manifesting in the ubiquitin-associated domain of p62 were associated with Paget’s disease of bone,mutations affecting all functional domains of p62 have now been identified in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.p62 is a multifunctional protein that facilitates protein degradation through autophagy and the ubiquitin-proteasome system,and also regulates cell survival via the Nrf2 antioxidant response pathway,the nuclear factor-kappa B signaling pathway and apoptosis.Dysfunction in these signaling and protein degradation pathways have been observed in amyotrophic lateral sclerosis and frontotemporal lobar degeneration,and mutations that affect the role of p62 in these pathways may contribute to disease pathogenesis.In this review we discuss the role of p62 in these pathways,the effects of p62 mutations and the effect of mutations in the p62 modulator TANK-binding kinase 1,in relation to amyotrophic lateral sclerosis-frontotemporal lobar degeneration pathogenesis.

Paterns and regularity of ring distribution of seismic activity before great earthquakes in China

A systematic study on ″ring phenomena″ frequently occurring before great earthquakes has made in this paper, which has analyzed the features of ring distributions before 16 great earthquakes and part of large earthquakes in China and its boundary areas, and discussed their features of generality, regularity and predictive meaning. The results have showed that moderate earthquakes or larger earthquakes distribute around the epicenter like a ring from decades to hundred years before the great earthquakes of magnitude more than 7, which is a general phenomenon of great earthquakes without an exception. The active ring generally occurs in the areas from hundreds to thousands of kilometers from the epicenter(according to the magnitude). The seismicity in the ring has three basic stages with different features. in the first stage, seismicity remains at low level and the earthquakes distribute scatteredly, while the source area of the future great earthquake remains quiet; in the second stage, the seismicity strengthens, whose frequency, intensity, concentrated degree, released rate of strain and ratio of distributed area etc. increase, while the quiet area decreases or disappears; in the third stage, the seismicity is weaker than in the former stage, and the quiet area appears again. The source area surrounded by the active ring might have three periods of activity(called as early term, medium term and late term foreshocks activity). The length of the quiet area undergoes the process from large to small, then to large. Therefore, we can estimate the occurring place, magnitude and seismogenic stage of great earthquake according to the area,length and the seismicity in the active ring, which is valuable to make a long term prediction of great earthquakes. At last, we had a preliminary discussion on the mechanism of active ring formation.

Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl β-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems em- ployed in earlier Src expression.

Primary Purification of Co-expressed Soluble and Insoluble Alpha-interferon 2b from Recombinant E. coli

Alpha-interferon 2b (IFN 2b) was produced both in soluble and insoluble forms from recombinant E. coli. The dissolution of the expressed IFN 2b in inclusion body was carried out and it was found that the optimal condition to dissolve the expressed protein was 7 mol稬1guanidinium salt solution at pH 3.0. The resultant solution was diluted 20 times using pH 6.0 buffer to refold the protein correctly. The cation exchange column was employed to purify both refolded and soluble IFN 2b. For soluble IFN sample, high IFN 2b recovery yield (92.1%) with 91.7% purity was obtained in the eluate. However, for refolded IFN sample, only 72.7% of IFN 2b was recovered with relatively low purity (56.8%) by cation exchange chromatography. Although the expression level of insoluble IFN was higher than that of co-expressed soluble IFN in this recombinant E. coli cells, the productivity of bioactive IFN 2b was higher with soluble expressed IFN after primary purification process. Soluble expression of foreign proteins in recombinant bacteria might be an alternative strategy for efficient production of heterogeneous pro-teins due to high bioactivity and simple downstream protein purification process.

Expression Characterization and Preparation of Human Amyloid Precursor Protein in Escherichia coli

To analyze whether expressed amyloid precursor protein（APP） existed in hydrophilic（cytoplasmid） or hydrophobic（lipid bilayer） environment in E. coli and to obtain intact APP for study on its function, we investigated the expression characterization and preparation of the three intact isoforms APP770, APP751, and APP695 in E. coll. The results show that these expressed APPs existed both in hydrophilic cytoplasm region as inclusion bodies and hydrophobic membrane region as membrane-bound state in E. coll. APPs in inclusion bodies were purified on an NTA-Ni^2＋ agarose column after dissolving in the urea buffer and APPs in membrane-bound state were obtained by ultracentrifugation. The activity analysis indicates that APP770 and APP751 exhibited strong trypsin-inhibitory activity like the natural ones. These results indicate that E. coli cells can be used as host cells for the expression of human integral membrane protein like APP in either soluble or membrane-bound state unless the interest protein undergone post-translational modification is required.

Expression of Fragaria ananassa Osmotin-like Protein(FaOLP2) in E. coli:Purification and Antifungal Activity

[ Objective] The FaOLtr2 （ Frago^ia ananassa osmotin-like protein） is a functional homolog of PR5-1ike protein. This study was undertaken to produce recombinant FaOLP2 and to identify its antifungal activity. [ Method] The ORF of FaOLP2 （ accession number DQ325524） was cloned into pET22b vector to con- stroct the pET22b-FaOLP2 plasmid. The recombinant mature FaOLP2 was expressed in E. coli Rosetta-gami B （DE3） by inducing with I nunol/L IPTG and found exclusively in insoluble inclusion bodies. As FaOLP2 requires the correct formation of eight disulfide bonds, but there were no obvious effect to correctly form these by expression at different temperatures and high osmotic pressure （ supplemented Betaineand and D-Sorbitol）, we used an in vitro method to refold E. coli expressed FaOLP2 by gradually elution using reduced：oxidized gluthatione redox buffer, followed by 8 mol/L urea solubilized His6-tagged mature FaOLP2 protein, which was affinity-purified by an immobilized-metal （Ni2＋ ） affinity chromatography （IMAC） column. [ Result] This method generated biologically active conformations of the recombinant mature FaOLP2 that displayed antifungal activity against Ustilaginoides virens, a plant pathogenic fungus, which causes rice false smut. [ Conclusion] This study laid the foundation for further biotechnological application of the novel protein.

Pathogenic and pathological characteristic of new type gosling viral enteritis first observed in China

AIM: To study the purifying method and characteristics of new gosling viral enteritis virus (NGVEV),the etiological agent of new gosling viral enteritis (NGVE) which was first recognized in China, as well as the pathomorphological development in goslings infected artificially with NGVEV. METHODS: (1)NGVEV virions were purified by the procedure of treatment with chloroform and ammonium sulfate precipitation, dialysis to remove the sulfate radical and ammonium ion and separation by gel filtration chromatography, and SDS-PAGE. (2)Forty-2-day-old White Sichuan goslings were orally administered with NGVEV and 24 hr later 2 birds were randomly selected and killed at 24hr intervals until death occurred. Specimens(duodenum, ileum, liver, heart, kidney, spleen, lung, proventriculus, pancreas, esophagus, and the intestinal embolus) were taken until all birds in this group died and were sectioned and stained with hemotoxylin and eosin and studied by light microscope. RESULTS: NGVEV shared the typical characteristics of Adenovirus and which structural proteins consisted of 15 polypeptides. Necrosis and sloughing of the epithelial cells covering the villus tips of the duodenum were first observed in goslings 2 days postinfection artificially with NGVEV. With the progress of infection, this lesion rapidly occurred in the epithelium at the base of the villus and with infiltration of the inflammatory cells, the jejunum tended to be involved. With the intensification of mucosa necrosis and inflammatory exudation of the small intestine, fibrinonecrotic enteritis was further developed and embolus composed of either intestinal contents wrapped by pseudo-membrane or of the mixture of fibrous exudate and necrotic intestinal mucosa were observed in the middle-lower part of the small intestine. This structure occluded the intestinal tract and made the intestine dilated in appearance. The intestinal glandular cells underwent degeneration, necrosis and might be found sloughed into the lumen. Hemorrhage and hyperemia could be observed on the lung and kidney. Epithelial cells of the renal tubular underwent degeneration. In some cases, granular degeneration and fatty degeneration could be found in the liver and in some cases at a later stage of this disease the epithelial cells of trachea and proventriculus might be found sloughed. In some cases at an early stage of this disease, cardiac hyperemia and hemorrhage could be observed. Esophagus, pancreas and brain were found normal. Analyses and comparisons between the pathologic lesions of NGVE and Gosling Plague (GP) were available in this paper as well. CONCLUSION: (1)NGVEV is adenovirus. (2)Pathological characteristic could be as the data for NGVE diagnosis.

On seismic strengthening area before strong and great shocks and its mechanism

By systematically studying seismic strengthening areas before 85 earthquakes with M>=6.0 in China, some results have been extracted. 1) Earthquake active strengthening area exists universally before strong shock or great earthquake; 2) The size of the strengthening area and its appearing time will increase when the earthquake magnitude increases; 3) The rate between the size of seismic strengthening area and the size of the source region decreases when earthquake magnitude increases; 4) The appearing time of the earthquake active strengthening region in the eastern part of China is longer than that in the western part of China. The above characteristics have been preliminarily explained qualitatively and half-quantitatively by applying the strong body earthquake generating model and the hard inclusion theory. Then applying the seismic strengthening area, we have obtained long-term predictions of 2 earthquakes, so the seismic strengthening area before strong earthquake or great earthquakes is a universal phenomenon, which has some mechanical base.

Tissue plasminogen activator-independent roles of neuroserpin in the central nervous system

A number of studies have confirmed the existence of tissue-type plasminogen activator-independent roles of neuroserpin, a member of the serine protease inhibitor superfamily. In this review article, we aim to clarify this role. These unique roles of neuroserpin are involved in its neuroprotective effect during ischemic brain injury, its regulation of tumorigenesis, and the mediation of emotion and cognition through the inhibition of urokinase-type plasminogen activator and fibrinolysin, modification of Th cells, reducing plaque formation, promoting process growth and intracellular adhesion, and alterina the expression of cadherin and nuclear factor kaooa B.

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.

hVEGF165 Expression in Escherichia coli Conserves Its Biological Function

The paper describes the expression of human protein VEGF165 in Escherichia coli and its purification. This growth factor isoform contains exon 7, which is essential for binding to extracellular domain of VEGF receptor 2, located on endothelial cells lining the surface of blood vessels. This binding stimulates the cascade of downstream signalling events leading to process known as angiogenesis, hVEGF165 overexpressed with His-tag in BL21 E. coli cells forms inclusion bodies （insoluble protein）, so the research found the procedure for its solubilization and purification on a Nickel based affinity chromatography. Although this eukaryotic signal protein needs posttranslational processing for its full function as a homodimer, author verified the biological activity of our hVEGF165 protein, obtained as monomer, by wound healing test.

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1.Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR),then cloned into vector pET32a(+),and expressed as thioredoxin-PedA fusion protein in the host strain E.coli BL21 (DE3).The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column.Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase.Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E.coli (pPA003PED1) using Listeria monocytogenes as the indicator strain.Thioredoxin-PedA fusion gene was further cloned into pET20b(+).Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces.The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E.coli (pPA003PED2).Therefore,biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.