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Yang S.,CAS Guangzhou Institute of Biomedicine and Health | Kuang Y.,CAS Guangzhou Institute of Biomedicine and Health | Kuang Y.,Jilin University | Li H.,CAS Guangzhou Institute of Biomedicine and Health | And 14 more authors.
PLoS ONE | Year: 2013

Pichia pastoris is one of the most widely used expression systems for the production of recombinant secretory proteins. Its universal application is, however, somewhat hampered by its unpredictable yields for different heterologous proteins, which is now believed to be caused in part by their varied efficiencies to traffic through the host secretion machinery. The yeast endoprotease Kex2 removes the signal peptides from pre-proteins and releases the mature form of secreted proteins, thus, plays a pivotal role in the yeast secretory pathways. In this study, we found that the yields of many recombinant proteins were greatly influenced by Kex2 P1' site residues and the optimized P1's amino acid residue could largely determine the final amount of secretory proteins synthesized and secreted. A further improvement of secretory yield was achieved by genomic integration of additional Kex2 copies, which again highlighted the importance of Kex2 cleavage to the production of recombinant secretory proteins in Pichia yeast. © 2013 Yang et al.


Hu Z.,Hunan Normal University | Hu Z.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Zhou X.,Hunan Normal University | Chen J.,Hunan Normal University | And 5 more authors.
Toxins | Year: 2014

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000-4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na+, K+ and Ca2+ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na+ currents and T-type Ca2+ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD50 was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom. © 2014 by the authors; licensee MDPI, Basel, Switzerland.


Hu Z.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Hu Z.,Hunan Normal University | Xiao Z.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Zhou X.,Hunan Normal University | And 3 more authors.
Chinese Journal of Chromatography (Se Pu) | Year: 2015

Selenocosmia jiafu (S.jiafu) is recently identified as a new species of spider in P. R. China. These medium bodied venomous spiders are distributed mainly in the hilly areas of southwest of China, mostly at Yunnan and Guangxi Provinces. In order to understand the composition of the S. jiafu venom, we performed a preliminary analysis of this venom using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser-desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The S. jiafu venom was separated by RP-HPLC in an analytical C18 column (phenomenex 100 Å, 250 mm × 4.6 mm, 5 μm) equilibrated with solution A (distilled water with 0.1% trifluoroacetic acid), using a gradient from 0% to 50% of solution B (acetonitrile with 0.1% trifluoroacetic acid) over 50 min with a flow rate of 1 mL/min. The isolated venom proteins were treated with in-gel digestion separated by SDS-PAGE and then identified by liquid chromatography-electrospray ionization quadrupole-time of flight mass spectrometry (LC-ESI-QTOF-MS) techniques. The results show that more than 40 fractions eluted were monitored at 215 nm in the RP-HPLC chromatogram of the venom of the spider S. jiafu. Most of the fractions were eluted with retention times of 5-15 min and 25-40 min, corresponding to 5%-15% and 25%-40% acetonitrile, respectively. The venom contains 238 peptides that follow a bimodal distribution, with about 62.5% of the peptides having a relative molecular mass of 3 000-4 500 and about 33.2% of the peptides having a relative molecular mass of 1 000-3 000. This distribution model is rather different from those of peptides from other tarantula spider venoms analyzed. To explore the relative molecular mass distribution of the venom proteins, the venom was analyzed by SDS-PAGE using standard protocols. Except for peptides with relative molecular mass lower than 10 000, the SDS-PAGE electrophoresis revealed three more obvious bands around 50, 72 and 90 kD respectively. Further MS analysis indicated that there are mainly hemocyanin, potassium ion channel protein, calcium protease and so on. Altogether, this study not only indicated there are many peptides and proteins in the S. jiafu venom, but also provided a basis for further case-by-case investigation of peptide toxins from this venom.


Sun W.,Chinese Academy of Sciences | Sun W.,Anhui University | Lai Y.,Chinese Academy of Sciences | Li H.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | And 10 more authors.
Journal of Immunological Methods | Year: 2016

Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75mg/L rhIL-15 after 3days of induction by methanol. The rhIL-15 was purified by Ni+-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from P. pastoris. © 2015 Elsevier B.V.


Li H.,CAS Guangzhou Institute of Biomedicine and Health | Li H.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Hui X.,University of Hong Kong | Yang S.,CAS Guangzhou Institute of Biomedicine and Health | And 11 more authors.
Protein Expression and Purification | Year: 2013

Platelet-derived growth factors (PDGFs) are important biochemical mediators regulating many physiological and pathophysiological processes, including promotion of the chemotactic recruitment and proliferation of cells involved in wound repair. Previously, homodimers of rhPDGF-AA protein were purified from Escherichia coli. However, eukaryotic proteins often contain posttranslational modifications, such as glycosylation, that are required for biological functions. In this study, an efficient method was established to purify a glycosylated rhPDGF-AA dimer from P. pastoris culture media by one step CM Sepharose ion exchange chromatography yielding about 20 mg/L of over 95% highly purified rhPDGF-AA. Mass spectrometry analysis of the purified rhPDGF-AA displayed a molecular weight (MW) of 27,825.513 Da, composed of a subunit with MW of 15,042.945 Da and a subunit with MW of 12,904.374 Da. The size difference is accounted for by differential glycosylation of the monomers. Biological activity of the rhPDGF-AA was confirmed by its ability to induce NIH/3T3 cells proliferation. The experimental procedure we have developed facilitates production of an active glycosylated rhPDGF-AA in large amounts for further research and drug development. © 2013 Elsevier Inc. All rights reserved.


Li H.-B.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Yang R.-X.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Chen J.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Li Y.-Y.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Hu X.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province
Chinese Traditional and Herbal Drugs | Year: 2014

Objective: To prepare the recombinant Poria cocos immunomodulatory protein-1 (WCFIP1) and its antibody. Methods: The cDNA of WCFIP1 was synthesized, cloned into plasmid and transformed into Escherichia coli expression strain. The recombinant protein of WCFIP1 was expressed, purified, and injected into mice to prepare antiserum. The antibody against WCFIP1 was purified by portein-G column and the specificity was determined by Western blotting. Results: The recombinant protein of WCFIP1 and the specific antibody were prepared. Conclusion: The recombinant protein of WCFIP1 is expressed and purified from E. coli, and the specifity of the prepared antibody is determined. ©, 2014, Editorial Office of Chinese Traditional and Herbal Drugs. All right reserved.


Yang X.,Huaihua College | Yang X.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Zhang F.,Huaihua College | Hu Y.,Huaihua College | And 4 more authors.
International Journal of Electrochemical Science | Year: 2014

An electrochemical sensor based on gold nanoparticals (Au) doping graphene sheets (GS) nanocomposites sensitized screen-printed carbon electrode (SPCE) as a disposable platform was developed for voltammetric determination of guaiacol (IUPAC name, 2-methoxyphenol) in bamboo juice. The SPCE was first modified by a dispersed solution of GS-Nafion (Nf), which was followed by electroless plating of Au on the surface to fabricate SPCE{box drawings light vertical}GS-Nf/Au electrode. X-ray powder diffractometer (XRD), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) were employed to characterize the synthesized GS and the construction processes of the modified electrode. The determination of guaiacol at the modified electrode was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in the phosphate buffer solution (PBS). Under the optimum experimental conditions, the peak currents obtained at +0.22 V from DPV were linearly dependent on the guaiacol concentrations in the range of 1.0×10-7 mol/L to 1.5×10-5 mol/L. The detection limit (3σ) was 5.4×10-8 mol/L. The proposed modified electrode is sensitive, rapid, simple and disposable. In addition, the proposed sensor showed satisfactory recovery results toward the commercial bamboo juice samples. © 2014 The Authors.


He G.,Hunan City University | Yang X.,Huaihua College | Yang X.,Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province | Hu Y.,Hunan City University | And 2 more authors.
International Journal of Electrochemical Science | Year: 2014

A sensitive and selective amperometric immunosensor for chloramphenicol (CAP, IUPAC name: 2, 2-Dichloro-N-[2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl) ethyl] acetamide) detection based on magnetic nanocomposites modify screen-printed carbon electrode (SPCE) as a disposable platform was fabricated. Graphene sheets (GS)-Nafion (Nf) dispersed solution was first dropped on the SPCE and then Fe3O4-Au nanoparticles (GoldMag particles, GMP) coated bovine serum albumin-CAP (BSA-CAP) conjugates was absorbed on it with the aid of external magnetic field. X-ray powder diffractometer (XRD), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM) were employed to characterize the synthesized GS and the construction processes of the modified electrode. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study its electrochemical properties. The content of CAP was determined with a competitive immunoassay mode. When different concentration of CAP and 1.0 μg/mL anti-CAP were added to the phosphate buffer solution (PBS) containing 2.0 mmol/L K3[Fe(CN)6], the increase ratio of the DPV current (CI%) was proportional to the concentration of CAP over the range from 2.0 ng/mL to 200.0 ng/mL after incubation for 5.0 min at 25 °C. The detection limit was 0.82 ng/mL (S/N=3). The immunosensor was employed to determine CAP in milk samples and the results were consistent with high-performance liquid chromatography (HPLC) method. The proposed amperometric immunosensor is sensitive, selective, rapid, magnetic field controllable, low sample consumable and disposable. Results obtained in this study demonstrate that the immunosensor was suitable for determining trace CAP in real samples. © 2014 The Authors.

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