Jian-Ning Liu

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.

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.

Lung endothelium targeting for pulmonary embolism thrombolysis.

Background—Pulmonary embolism occurs frequently in hospitalized patients. Thrombolytic therapy, currently used as the major treatment, has often been associated with severe bleeding complications and has thereby been life-threatening. We have developed a novel therapeutic method based on our newly created pulmonary endothelium-specific antibody. Methods and Results—We isolated membrane proteins of rat pulmonary vascular luminal endothelium and obtained a monoclonal antibody, RE8F5, which antigen was uniquely expressed by the pulmonary capillary endothelium. In vivo biodistribution showed that RE8F5 and its urokinase conjugate were rapidly and specifically accumulated in lung. Urokinase and the conjugate were compared in rats with pulmonary, hepatic, and lower-limb embolus. In a pulmonary embolus model, the conjugate exhibited 12-fold enhanced thrombolytic potency over urokinase, whereas plasma fibrinogen and bleeding time were unaffected. In 2 other models, no significant thrombolysis was induced by the conjugate. In contrast, thrombolysis by urokinase was found to be comparable to the pulmonary embolus model. In addition, urokinase caused significant consumption of fibrinogen in all experiments. Conclusions—These data show that urokinase equipped with lung endothelium-specific antibody is an ideal treatment for pulmonary embolism, with a high efficacy of thrombolysis and low risk of bleeding.

Expression and characterization of recombinant human micro-plasminogen

Micro-plasminogen (μPlg) gene amplified from human liver cells by reverse transcription PCR was inserted into expression vector pET-28a (pET-28a/μPlg) and transformed into E. coli strain BL21(DE3). Recombinant human micro-plasminogen (rh-μPlg) was over-expressed as inclusion bodies when induced with IPTG. After renaturation and purification, 16xa0mg rh-μPlg/l was obtained with a homogeneity of 95% (w/w). Pro-urokinase (proUK)-induced rh-μPlg activation was significantly faster than when Glu-plasminogen was the substrate. The catalytic efficiency of urokinase (UK) activation of rh-μPlg was twice that of Glu-plasminogen. While recombinant human micro-plasmin (rh-μPlm) and Lys-plasmin had a similar amidolytic activity against a small substrate, d-valyl-l-leucyllysine-p-nitroaniline dihydrochloride, Lys-plasmin activated proUK with a catalytic efficiency about fourfold greater than did rh-μPlm. These results suggested that the kringle 1–5 domain of plasminogen and plasmin may modify both UK activation of plasminogen and plasmin activation of proUK, respectively.

Expression, purification and characterization of aprotinin and a human analogue of aprotinin.

Aprotinin is a Kunitz-type inhibitor with a relatively broad specificity. It has been shown to be clinically useful for the management of hemorrhagic complications. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant aprotinin and a human aprotinin analogue (cloned form human cDNA library). Both fusion proteins were overexpressed mainly as inclusion bodies in Escherichia coli and accounted for approximately 28% of the total cell proteins. After purification by Ni-Sepharose affinity chromatography and renaturation, the fusion proteins were cleaved with SUMO protease 1. Aprotinin and its analogue were separated from the fusion partner by the subtractive chromatography using Ni-Sepharose and then further purified with CM-cellulose. Kinetic studies demonstrated that the amidolytic activity of plasmin was competitively inhibited by recombinant aprotinin with a K(i) of 8.6+/-2.4 nM, which was similar to the K(i) (7.5+/-2.7 nM) of natural aprotinin. The K(i) of human aprotinin analogue was 22.7+/-6.5 nM. The expression strategy described in this study allows convenient high yield and easy purification of small recombinant protease inhibitors with complete native sequences.

Vaccination with DKK1-derived Peptides Promotes Bone Formation and Bone Mass in an Aged Mouse Osteoporosis Model

The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22xa0weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.

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.

Construction and characterization of a novel chimeric antibody c3C7 specific for the integrin αIIbβ3 complex

A murine monoclonal antibody (mAb) 3C7 against integrin αIIbβ3 was previously obtained as a potential antithrombotic agent in our laboratory. The epitope of 3C7 is a specific conformation of the αIIbβ3 complex, but not either of the two subunits, which makes it different from abciximab, a supplementary antibody drug used in percutaneous coronary intervention which has a cross-reaction with other integrins sharing the β3 subunit. To reduce the human anti-mouse antibody reactions of 3C7, the variable regions of this antibody were cloned and fused with the constant counterparts of human IgG1. Two vectors of light and heavy chains were constructed and co-transfected into CHO-dhfr− cells. The chimeric antibody c3C7 was purified and the properties of c3C7 were compared with 3C7. Identical to its parent antibody 3C7, c3C7 binds to the αIIbβ3 complex, but not to either of the subunits. The Kd value of c3C7 was in the same order of magnitude as 3C7 (1.570u2009±u20090.326 vs 0.780u2009±u20090.182xa0nmol/L). Human platelet aggregation induced by adenosine diphosphate was effectively inhibited by c3C7 in a dose-dependent manner. In conclusion, after the modification, c3C7 retained the properties of its parent mAb with no loss of its biological activity. Therefore, c3C7 has the potential to become a novel agent for the treatment of thrombosis.