Yanchun Tang

Chemical conjugation of urokinase to magnetic nanoparticles for targeted thrombolysis

Thrombolytic therapy is an important treatment for thrombosis, a life-threatening condition in cardiovascular diseases. However, the traditional thrombolytic therapies have often been associated with the risk of severe bleeding. By conjugating urokinase with magnetic nanoparticles (MNPs), we have presented a strategy to control thrombolysis within a specific site. The covalent bioconjugate of urokinase and dextran-coated MNPs was synthesized and isolated. Thrombolysis by the conjugate was studied under a magnetic field in a rat arteriovenous shunt thrombosis model. The magnetic field was generated by two AlNiCo permanent magnets around the site of thrombus. The magnetic field enhanced the thrombolytic efficacy of the conjugate by 5-fold over urokinase with no reduction in plasma fibrinogen and little prolonged bleeding time. It suggested that thrombolysis had been specifically directed to the desired site by the magnetic carrier under the magnetic field. Additionally, the conjugate had a longer half-life than urokinase in circulation.

Biofunctionalisation of porous silicon (PS) surfaces by using homobifunctional cross-linkers

Porous silicon surfaces can be bio-functionalised by a simple three-step method. First the hydrogen-terminated porous silicon was oxidised and amino-silanised in a one-pot reaction by 3-aminopropyl(triethoxyl)silane with the aid of an organic base, diisopropylethylamine. Secondly, the primary amine reacted with either of two homobifunctional cross-linkers, bis(N-succinimidyl)carbonate and (N,N′-bis(p-maleimidophenyl)methylene. By modulating the reaction conditions, a high surface coverage of linking groups, succinimidyl ester or maleimide, can be obtained separately. Since homobifunctional cross-linkers can form bridged structures with both ends fixed to the surface, the reaction conditions were optimised for one end attached to the surface and the other end pendent. Succinimidyl ester is an amino-reactive group, therefore mouse monoclonal antibody bearing amino groups was grafted. An enzyme linked immunosorbent assay was used to evaluate the surface density of antibody at 0.008 ng cm−2. The other linker, (N,N′-bis(p-maleimidophenyl)methylene, was refluxed in acetonitrile with surface amines to result in maleimde-terminated surfaces. Then a reduced urokinase bearing accessible thiol groups was grafted and its enzymatic activity was assayed at 0.004 nmol cm−2 for urokinase. Transmission infrared, X-ray photoelectron, interferometric reflectance, UV-Vis, photoluminescence spectroscopies and a chromogenic assay were used to characterise the surfaces.

Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage.

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.

Split Ssp DnaB mini‐intein‐mediated production of recombinant human glucagon‐like peptide‐1/7‐36

Glucagon‐like peptide‐1 (GLP‐1) plays an important role in the regulation of postprandial insulin release. Here, we used the split DnaB mini‐intein system to produce recombinant human GLP‐1/7‐36 (rhGLP‐1) in Escherichia coli. The C‐terminal domain of DnaB mini‐intein (IntC) was genetically fused at the N‐terminus of rhGLP‐1 to produce IntC–GLP‐1. IntC–GLP‐1 and N‐terminal domain of DnaB mini‐intein (IntN) protein were prepared in a denatured buffer of pH 8.0. IntC–GLP‐1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC–GLP‐1 at the molar ratio of 1:2. Then, rhGLP‐1 was released from IntC–GLP‐1 via inducible C‐terminal peptide‐bond cleavage by shifting pH from 8.0 to 6.6 at 25 °C for 24‐H incubation. Then, the supernatant was applied to a Ni‐Sepharose column, and the pass through fraction was collected. About 5.34 mg of rhGLP‐1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3,300.45 Da, which was equal to the theoretical value of GLP‐1/7‐36. The glucose‐lowering activity of rhGLP‐1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP‐1 without using enzyme or chemical reagents, which could also be used for other similar peptides.

The Novel Pro-Osteogenic Activity of NUCB21–83

NUCB21–83 has been recently reported as an anorexigenic and anti-hyperglycemic peptide. Here we report that NUCB21–83 promotes osteogenesis. It was found after two months of once-a-day intravenous injection of NUCB21–83, bone mineral density of femora and lumbar vertebrae were increased in ovariectomized rats. NUCB21–83 also increased the alkaline phosphatase activity and promoted mineralization in mouse MC3T3-E1 preosteoblastic cell line. When either both Arg60 and Arg63 or Ser72 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 NUCB21–83 itself. These findings warranted that further studies on NUCB21–83 would be valuable for the treatment of bone metabolic diseases especially for osteoporosis.

Expression, purification, and characterization of mouse nesfatin‐1 in Escherichia coli

Nesfatin‐1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short‐term and long‐term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin‐1 were cloned into a pET28a vector after the hexa‐histidine‐tagged multiple cloning sites sequence with an enterokinase recognition site incorporated in‐between. The recombinant plasmid was transformed into Escherichia coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins. After purified by Ni‐sepharose affinity chromatography, the fusion protein was treated with enterokinase to release nesfatin‐1. The nesfatin‐1 sample was further purified with reverse‐phase high performance liquid chromatography (HPLC), and its molecular weight was determined by mass spectrometry. The biological activities of recombinant nesfatin‐1 were also assessed using in vivo animal models. The method described here promises to produce about 8 mg biologically active nesfatin‐1 with homogeneity over 98% from 1‐L shaking flask culture of E. coli, which can be considered as an easy and cost‐effective way to synthesize nesfatin‐1.

Expression, purification and characterization of mouse nesfatin-1 in E. coli.

Nesfatin‐1 is a newly discovered satiety molecule expressed mainly in the hypothalamic nuclei. It suppresses both short‐term and long‐term appetite. Six synthetic deoxyoligonucleotides overlapped by PCR encoding nesfatin‐1 were cloned into a pET28a vector after the hexa‐histidine‐tagged multiple cloning sites sequence with an enterokinase recognition site incorporated in‐between. The recombinant plasmid was transformed into Escherichia coli strain Rosetta to express the fusion protein, which constituted 27% of the total cell proteins. After purified by Ni‐sepharose affinity chromatography, the fusion protein was treated with enterokinase to release nesfatin‐1. The nesfatin‐1 sample was further purified with reverse‐phase high performance liquid chromatography (HPLC), and its molecular weight was determined by mass spectrometry. The biological activities of recombinant nesfatin‐1 were also assessed using in vivo animal models. The method described here promises to produce about 8 mg biologically active nesfatin‐1 with homogeneity over 98% from 1‐L shaking flask culture of E. coli, which can be considered as an easy and cost‐effective way to synthesize nesfatin‐1.

OLIGONUCLEOTIDE-HIRULOG18 CONJUGATED BY CLICK CHEMISTRY AND ITS INHIBITION ON THROMBIN

We have shown previously that oligonucleotide conjugated hirulog (bivalirudin) has a higher inhibitory activity against thrombin compared with hirulog. The negative charged oligonucleotide was considered to be responsible for the additive activity. To further investigate the related effect of oligonucleotide, the oligonucleotide conjugated hirulog18 was prepared in the present work and the activity of the conjugate against thrombin was measured. Hirulog18, a peptide lack of two N-terminal amino acid residues of hirulog, has little inhibitory activity against thrombin compared with hirulog. The conjugate oligonucleotide-hiruglog18 was successfully synthesized using click chemistry and validated by MALDI-MS and gel electrophoresis. The activity of the conjugate against thrombin was measured by the chromogenic assay using Chromozym TH as substrate. It was found surprisingly that oligonucleotide-hirulog18 had an inhibitory effect better than hirulog18 and hirulog. Strong negative charged heparin was used to study the binding mode between oligonucleotide-hirulog18 and thrombin. The results suggested that the negative charged oligonucleotide could be helpful for the conjugates binding to the anion-binding exosite of thrombin via the Coulomb force. The highly inhibitory effect of the oligonucleotide-hirulog18 conjugate against thrombin may present a new strategy to generate a novel class of direct