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Ruishu Li,
Qiong Wu,
Yue Zhao,
Wenbo Jin,
Xinfang Yuan,
Xiaopeng Wu, Yanchun Tang,
Jing Zhang,
Xiangyang Tan,
Feng Bi,
Jian-Ning Liu
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ABSTRACT: NUCB2(1-83) has been recently reported as an anorexigenic and anti-hyperglycemic peptide. Here we report that NUCB2(1-83) promotes osteogenesis. It was found after two months of once-a-day intravenous injection of NUCB2(1-83), bone mineral density of femora and lumbar vertebrae were increased in ovariectomized rats. NUCB2(1-83) also increased the alkaline phosphatase activity and promoted mineralization in mouse MC3T3-E1 preosteoblastic cell line. When either both Arg(60) and Arg(63) or Ser(72) were mutated to Ala, the pro-osteogenic activity was completely lost, indicating that these residues are structurally important for its biological function. Furthermore, it encumbered osteoclastic differentiation of RAW 264.7 macrophage. It also excluded any possibility of the effect caused by contaminants or experimental faults, and demonstrated that the pro-osteogenic activity observed was a specific effect of NUCB2(1-83) itself. These findings warranted that further studies on NUCB2(1-83) would be valuable for the treatment of bone metabolic diseases especially for osteoporosis.
PLoS ONE 01/2013; 8(4):e61619. · 4.09 Impact Factor
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ABSTRACT: Purification tags are robust tools that can be used to purify a variety of target proteins. However, tag removal remains an expensive and significant issue that must be resolved. Based on the affinity and the trans-splicing activity between the two domains of Ssp DnaB split-intein, a novel approach for tag affinity purification of recombinant proteins with controllable tag removal by inducible auto-cleavage has been developed. This system provides a new affinity method and avoids premature splicing of the intein fused proteins expressed in host cells. The affinity matrix can be reused. In addition, this method is compatible with his-tag affinity purification technique. Our methods provide the insights for establishing a novel recombinant protein preparation system.
Journal of chromatography. A 03/2011; 1218(18):2553-60. · 4.19 Impact Factor
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ABSTRACT: Nesfatin-1 is recently reported as a satiety molecule to suppress food intake via the melanocortin signaling in hypothalamus when injected centrally and peripherally. Here we report that nesfatin-1 is also anti-hyperglycemic. It was found that the intravenous injection of nesfatin-1 significantly reduced blood glucose in hyperglycemic db/db mice. This anti-hyperglycemic effect of nesfatin-1 was time-, dose-, insulin-dependent and peripheral.
Biochemical and Biophysical Research Communications 12/2009; 391(1):1039-42. · 2.48 Impact Factor
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ABSTRACT: Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.
Protein Expression and Purification 11/2007; 55(2):312-8. · 1.59 Impact Factor
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ABSTRACT: The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted to pET-29a and expressed in E. coli Rosseta. After purification by nickel-affinity and refolding, this scFv protein was proven to recognize reteplase specifically and sensitively in ELISA and dot-blotting. Its binding constant to reteplase was 1.84x10(-8) M, measured by surface plasmon resonance. After immobilized on Sepharose 4B, the scFv was used for the affinity purification of reteplase from milk. It was found that reteplase was highly purified from the starting material. In conclusion, it has been demonstrated that humanized scFv prepared with this approach could be used as a practical affinity ligand for efficient and cost-effective purification of reteplase, as well as other therapeutic proteins.
Journal of Biochemical and Biophysical Methods 05/2006; 67(1):27-36. · 2.33 Impact Factor
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ABSTRACT: An anti-E. coli thioredoxin monoclonal antibody, IMM-3C6, which showed high specificity to thioredoxin as assessed by indirect ELISA, was generated using hybridoma technology. The affinity constant of IMM-3C6 to thioredoxin was 0.40 x 10(9) M: (-1) and its sensitivity to thioredoxin fusion protein in dot blotting was 50 ng. In sandwich ELISA, it detected thioredoxin fusion protein between 16 and 150 ng/ml. By using IMM-3C6 as the ligand, thioredoxin fusion protein was successfully purified by affinity chromatography. IMM-3C6 was confirmed to be a useful tool for immunoassay and purification of thioredoxin fusion proteins.
Biotechnology Letters 03/2006; 28(3):183-8. · 1.68 Impact Factor
