重组内皮抑素的性质表征与生物活性分析

通过SDS—PAGE电泳、基质辅助激光解吸电离飞行时间质谱、高效液相色谱、地高辛方法、蛋白印迹方法、鸡胚尿囊膜血管生成抑制实验和透射电子显微镜法对重组内皮抑素进行纯度分析、鉴定和生物活性研究。重组内皮抑素纯度达到99％，能够抑制鸡胚尿囊膜新血管生成和人脐静脉内皮细胞ECV304增殖，引发内皮细胞的凋亡。     

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via alphavbeta3 and alpha5beta1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.     

Development of a novel reporter gene vector for cell based angiogenic studies

Angiogenesis is a promising area of research that targets key therapeutic areas like cancer; wound healing, inflammatory diseases, etc. There is an increasing demand for screening of potential angiogenic and anti-angiogenic agents using sensitive, robust cell-based assays. We have developed a reporter vector containing cis-acting elements that respond to growth factors/angiogenic ligands for use in a cell-based luciferase reporter assay. We performed transient transfection of our reporter gene vector in MCF-7 cells to establish its application for screening of potential pro/anti-angiogenic agents. Reporter gene transactivation studies with different concentrations of fetal bovine serum clearly indicated that the vector is functionally responsive to the angiogenic signals mediated by serum growth factors. We also used endostatin to inhibit transactivation and prove responsiveness to the anti-angiogenic agent. This vector is a promising tool for studying angiogenesis using cell-based reporter gene assays.     

重组内皮抑素的质谱分析及对内皮细胞周期的影响

采用质谱、圆二色谱、荧光光谱与细胞周期分析技术研究了重组内皮抑素的蛋白结构和对内皮细胞的作用机制.研究发现,以包涵体方式表达的蛋白中会有未被降解的N末端甲硫氨酸产物,重组内皮抑素在G₂期阻断内皮细胞的生长.表明内皮抑素引发内皮细胞凋亡与细胞周期相关,为抗血管生成治疗肿瘤研究奠定了实验基础.     

稀土离子对重组内皮抑素与肝素相互作用的影响

通过荧光光谱法和圆二色谱法研究了重组内皮抑素与稀土离子和肝素的作用.结果表明,稀土离子和肝素都可以与重组内皮抑素结合并引起蛋白的荧光猝灭和二级结构的改变,稀土离子与重组内皮抑素的结合会影响其与肝素的相互作用,并降低重组内皮抑素对人脐静脉内皮细胞的增殖抑制活性.     

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.     

Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometry.

Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.     

Chemical modification of fumagillin. I. 6-O-acyl, 6-O-sulfonyl, 6-O-alkyl, and 6-O-(N-substituted-carbamoyl)fumagillols.

The hydroxy group of fumagillol (3), a degradation product of fumagillin (1), was acylated, sulfonylated, alkylated or carbamoylated, and the anti-angiogenic activity of the resulting products was examined. These compounds inhibited the angiogenesis induced by basic fibroblast growth factor in the rat corneal micropocket assay and the growth of vascular endothelial cells in vitro. Among them, compound 2 (AGM-1470) was found to show the most potent inhibitory effect on the growth of vascular endothelial cells and was selected from this series as a candidate for further development.     

Cryptotanshinone but not tanshinone IIA inhibits angiogenesisin vitro

In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone from Salvia miltiorrhiza was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. Tanshinone IIA, another tanshinone isolated from S. miltiorrhiza, which is structurally very similar to cryptotanshinone except C-15 position of dihydrofuran ring does not inhibit angiogenesis induced by bFGF. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydrofuran ring plays a crucial role in the activity.     

Industrial case study: Evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli

Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.     

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.     

Expression and Purification of Soluble Porcine Cystatin 11 in Pichia pastoris

Cystatin 11 (CST11) belongs to the cystatin type 2 family of cysteine protease inhibitors and exhibits antimicrobial activity in vitro. In this study, we describe the expression and purification of recombinant porcine CST11 in the Pichia pastoris system. We then assess its antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis by liquid growth inhibition assay. Kinetic studies indicate that the recombinant porcine CST11 has high potency against E. coli and S. aureus. Scanning electronic microscope analysis showed that CST11 might be targeting the bacterial membrane and, thus, could potentially be developed as a therapeutic agent for inhibiting microbe infection without the risk of antibiotic resistance.     

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.     

Effector-assisted refolding of recombinant tissue-plasminogen activator produced in escherichia coli

Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.     

MALDI-TOF-MS法对重组人内皮抑素蛋白分子质量测定

肿瘤的生长依赖于血管的生成,新生血管不仅为肿瘤生长提供必需的营养物质,而且为肿瘤细胞扩散提供了重要的途径。1997年哈佛大学的O'Reilly等发现了一种内源性新血管生成抑制因子内皮抑素(Endoscatin),显示出特异抑制激活的血管内皮细胞增殖和肿瘤新血管生成的生物学活性,其抗肿瘤作用具有高效、低毒、无耐药性的优点。目前,内皮抑素的研究引起了国内外广泛的兴趣,在美国已进行以安全性为目的的I期临床实验,国内也有多家公司对内皮抑素进行了抗肿瘤研究并申报一类新药。内皮抑素有望成为医治肿瘤而又没有化疗和放疗的毒副作用的一种新的治疗方法,但是否能作为药物应用于临床,尚需对内皮抑素的结构特点及抑制肿瘤和内皮细胞的作用机制等方面进行许多深入的研究。     

Recombinant Production and Characterization of SAC,the Core Domain of Par-4, by SUMO Fusion System

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137–195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.     

Fusarielin A as an anti-angiogenic and anti-proliferative agent: basic biological characterization

Fusarielin A shows anti-angiogenic activity in the human umbilical vein endothelial cell (HUVEC) tube formation assay. Structural development studies indicated the importance of the hydroxyl groups in this molecule. A 3H-labeled derivative and a fluorescent affinity-labeling agent were prepared and used to examine the cellular distribution and biological behavior of fusarielin A.     

Crystal Structure of a CD163 Scavenger Receptor Cysteine-rich Domain Produced in Pichia Pastoris

CD163 is an endocytic receptor specifically expressed in monocytes/macrophages and belongs to class B of the scavenger receptor cysteine-rich(SRCR) superfamily(SRCR-SF). It plays critical roles in various physiological and pathological pathways. In this study, the fifth SRCR domain(SRCR5) of porcine CD163(pCD163) was expressed in Pichia pastoris system. The recombinant protein was homogeneous after purification by metal-affinity and size-exclusion chromatography. The purified protein was then assayed for its biological function. Single crystals of pCD163 SRCR5 were obtained under the condition of 25% PEG 4000, 0.1 M citric acid pH_3.5 and its structure was finally determined at 1.80 ?. All these results provide functional pCD163 SRCR5 in large quantity for future research, and enrich the structural details for CD163 and the SRCR-SF.     

Epoxyquinol A, a highly functionalized pentaketide dimer with antiangiogenic activity isolated from fungal metabolites

A unique pentaketide dimer structure of a novel fungal metabolite with antiangiogenic activity, designated as epoxyquinol A (1), was determined on the basis of NMR spectral data as well as the X-ray crystallographic analysis. 1 inhibits the endothelial migration induced by vascular endothelial growth factor (ED100 = 3 mug/mL).     

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Grampositive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-α expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin^®, 25 mg of pure nisin with an activity of 1×10⁶ AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1∶1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.