Affinity Gel Research Articles

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

Lectins of <i>Erythrina poeppigiana</i> and <i>Erythrina steyermarkii</i> (Leguminosae): charaderization and mitogenic effect

Las diferentes especies de Erythrina se encuentran ampliamente distribuidas en Costa Rica y se las conoce popularmente con el nombre de "poró". En el presente estudio, se seleccionaron dos especies: Erythrina poeppigiana y Erythrina steyermarkii. Se prepararon extractos de las semillas en solución tampón salina de fosfatos y se verificó la presencia de lectinas en ellos mediante la técnica de hemaglutinación, utilizando eritrocitos humanos. Se trató de demostrar un efecto selectivo de la hemaglutinación empleando eritrocitos de varias especies de mamíferos, específicamente de carnero, caballo y conejo. Solo los eritrocitos de conejo fueron aglutinados con la lectina de E. steyermarkii. El efecto hemaglutinante de las dos lectinas fue inhibido con los siguientes carbohidratos: D-galactosa, N-D-acetil galactosamina, D-Iactosa y Drafinosa. La lectina de E. steyermarkii también fue inhibida con L-rhamnosa. Las dos lectinas fueron aisladas por filtración en gel y cromatografia de afinidad, usando lactosa como ligando. Las fracciones que resultaron positivas se analizaron mediante la técnica de electroforesis en gel de poliacrilamida y duodecil sulfato de sodio (SDSPAGE). Con la filtración en gel y el SDS-PAGE, se comprobó que las dos lectinas tienen una masa molecular aparente de 50 kDa y. que están formadas por dos subunidades de 25 kDa, aproximadamente. Se buscó un efecto mitogénico en las dos lectÍnas y se encontró que sólo E. steyermarkii lo manifestaba sobre células mononucleares humanas aisladas de sangre periférica. Se determinó la estabilidad de las ¡eetinas en diferentes ámbitos de temperatura (4 a 100°C) y de pH (2 a 12 ). Las dos lectinas se mantuvieron estables en un rango de temperatura de 40 a 70°C y en un pH de 2 a 10.

Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential.

An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest.

A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide.

The entire stx1 region from Escherichia coli O157:H7, containing two open reading frames (stx1a and stx1b), was cloned into pET-32a with a single promoter. This region was transformed into E. coli TransB (DE3), which is a trxB and gor mutation strain. After expression in the E. coli periplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD50 ) and median lethal dose (LD50 ) values of approximately 30 ng and 1.5µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3mg/L by one-step nickel affinity gel column chromatography. This method is an easy approach to the large-scale preparation of Stx1 at a reasonable cost.

Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 μM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.

Expression and purification of DNA binding domain of NR4A1

To express and purify NR4A1-DNA binding domain (DBD) protein of nuclear receptors. The fusion protein PET28a-NR4A1-DBD was constructed and purified with the nickel affinity chromatography, cation-exchange chromatography and gel filtration chromatography. The protein PET28a-NR4A1-DBD was mostly soluable at 24 °C. A total of 2-3 mg/L pure NR4A1 proteins were yielded in bacterial culture and the purity for final fractions of NR4A1-DBD protein were great than 95% by SDS-PAGE analysis. Nickel affinity chromatography is effective to purify protein. The protein purity can be further improved by the following methods including cation-exchange chromatography and gel filtration chromatography.

Surface binding sites in amylase have distinct roles in recognition of starch structure motifs and degradation

Carbohydrate converting enzymes often possess extra substrate binding regions that enhance their activity. These can be found either on separate domains termed carbohydrate binding modules or as so-called surface binding sites (SBSs) situated on the catalytic domain. SBSs are common in starch degrading enzymes and critically important for their function. The affinity towards a variety of starch granules as well as soluble poly- and oligosaccharides of barley α-amylase 1 (AMY1) wild-type and mutants of two SBSs (SBS1 and SBS2) was investigated using Langmuir binding analysis, confocal laser scanning microscopy, affinity gel electrophoresis and surface plasmon resonance to unravel functional roles of the SBSs. SBS1 was critical for binding to different starch types as Kd increased by 7–62-fold or was not measurable upon mutation. By contrast SBS2 was particularly important for binding to soluble polysaccharides and oligosaccharides with α-1,6 linkages, suggesting that branch points are key structural elements in recognition by SBS2. Mutation at both SBS1 and SBS2 eliminated binding to all starch granule types tested. Taken together, the findings indicate that the two SBSs act in concert to localize AMY1 to the starch granule surface and that SBS2 works synergistically with the active site in the degradation of amylopectin.

Structural insight into equine lentivirus receptor 1.

Equine lentivirus receptor 1 (ELR1) has been identified as a functional cellular receptor for equine infectious anemia virus (EIAV). Herein, recombinant ELR1 and EIAV surface glycoprotein gp90 were respectively expressed in Drosophila melanogaster S2 cells, and purified to homogeneity by Ni-NTA affinity chromatography and gel filtration chromatography. Gel filtration chromatography and analytical ultracentrifugation (AUC) analyses indicated that both ELR1 and gp90 existed as individual monomers in solution and formed a complex with a stoichiometry of 1:1 when mixed. The structure of ELR1 was first determined with the molecular replacement method, which belongs to the space group P42 21 2 with one molecule in an asymmetric unit. It contains eight antiparallel β-sheets, of which four are in cysteine rich domain 1 (CRD1) and two are in CRD2 and CRD3, respectively. Alignment of ELR1 with HVEM and CD134 indicated that Tyr61, Leu70, and Gly72 in CRD1 of ELR1 are important residues for binding to gp90. Isothermal titration calorimetry (ITC) experiments further confirmed that Leu70 and Gly72 are the critical residues.

Selective purification of intestinal maltase complex by affinity chromatography employing an uncompetitive inhibitor as the ligand

Affinity chromatography of specific enzymes is limited by the presence of related enzymes and the limited selectivity of the affinity ligand. We synthesized and investigated the use of an uncompetitive inhibitor as an affinity ligand, to leverage its three-component interactions. Use of the potent α-glucosidase uncompetitive inhibitor 2-aminoresorcinol as the ligand of the affinity gel offered selective purification of maltase-glucoamylase complex from the crude mixture of intestinal α-glucosidases.

A Glucosamine-Specific Lectin from Green Dragon No. 8 Beans (Phaseolus vulgaris) Induced Apoptosis on Nasopharyngeal Carcinoma Cells.

A lectin exhibiting antiproliferative activity on tumor cell lines but devoid of antifungal activity has been purified from Phaseolus vulgaris cv. Green Dragon no. 8 seeds. The lectin was a 60 kDa dimeric protein with two 30 kDa subunits. It was a glucosamine-specific lectin as implied from the inhibitory effect of glucosamine on hemagglutinating activity of the lectin. The steps for isolation of the lectin involved Affi-gel blue gel (affinity gel), Mono Q (anion exchanger), and Superdex 75 column (size exclusion). The lectin was purified 20.8-fold from the crude extract of the beans. The purified lectin showed antiproliferative activity on breast cancer MCF7 cell line and nasopharyngeal cancer HONE1 and CNE2 cell lines, but a low activity on normal skin fibroblast HSF98 cell line. The lectin was shown to induce apoptosis on HONE1 cells, as indicated by increased phosphatidylserine externalization and mitochondrial depolarization. It also blocked HONE1 cell division and kept the cells at the G2/M phase of the cell cycle.

Isolation and characterization of recombinant murine Wnt3a

Wnt proteins are a family of morphogens that possess potent biological activity. Structure–function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt–cyclodextrin complexes may facilitate structure–activity studies in the absence of adverse detergent effects.

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme

In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. l-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-l-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-l-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants KM and VMax for BTH were determined by using hyaluronic acid as a substrate. KM and VMax values obtained from the Lineweaver–Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.

Structural Analysis of Glucuronoxylan-specific Xyn30D and Its Attached CBM35 Domain Gives Insights into the Role of Modularity in Specificity*

Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/β)8 barrel with an associated β-structure, whereas the attached CBM35 displays a jellyroll β-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu(129) makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed.

Purification and Characterization of Carbonic Anhydrase from Ağrı Balık Lake Trout Gill (Salmo trutta labrax) and Effects of Sulfonamides on Enzyme Activity

Carbonic anhydrase (CA) was purified from Ağrı Balık Lake trout gill (fCA) by affinity chromatography on a sepharose 4B-tyrosine-sulfanilamide column. The fCA enzyme was purified with about a 303.9 purification factor, a specific activity 4130.4 EU (mg-protein)(-1), and a yield of 79.3 by using sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was found to be about 29.9 kDa. The kinetic parameters, K(M) and V(max) were determined for the 4-nitrophenyl acetate hydrolysis reaction. Some sulfonamides were tested as inhibitors against the purified CA enzymes. The Ki constants for mafenide (1), p-toluenesulfonamide (2), 2-bromo-benzene sulfonamide (3), 4-chlorobenzene sulfonamide (4), 4-amino-6-chloro-1-3 benzenedisulfonamide (5), sulfamethazine (6), sulfaguanidine (7), sulfadiazine (8), and acetozazolamide (9) were in the range of 7.5-108.75 μM.

Inhibition of DUSP13B Phosphatase Activity by PTP Inhibitor V

We next confirmed the inhibition mechanism of PTP inhibitor V on DUSP13B by kinetic measurements based on Michaelis-Menten equation. The Lineweaver-Burk plots show that the maximum reaction velocity (Vmax) is constant regardless of the presence of the inhibitor. This result indicates that PTP inhibitor V binds to the active site of DUSP13B and functions as a competitive inhibitor of DUSP13B (Fig. 2(b)). The calculated inhibition constant (Ki) value was 6.36 μM. To clarify the inhibitory effect of PTP inhibitor V on intact DUSP13B expressed in the mammalian cells, human embryonic kidney 293 (HEK 293) cells were transfected with FLAG-tagged DUSP13B expression plasmid and incubated for 48 h. Then the cells were harvested and lysed with PTP lysis buffer. FLAG-DUSP13B was immunoprecipitated from cell lysates by anti-FLAG M2 affinity gel. Immunoprecipitated DUSP13B was incubated with or without PTP inhibitor V and the phosphatase activities of DUSP13B were measured by using 3-O-methylfluorescein phosphate (OMFP) as a substrate. Since PTP inhibitor V is hardly soluble in Dulbecco's modified Eagle's medium (DMEM), we decided to treat the inhibitor to the immunoprecipitated DUSP13B. The phosphatase activity of DUSP13B decreased as the

Fractionation and identification of Alaska pollock skin collagen-derived mineral chelating peptides

Peptides with the ability to chelate dietary minerals have been reported to have potential as functional food ingredients. A collagen tryptic hydrolysate (CTH), previously shown to chelate iron, was further investigated for the presence of Ca, Fe and Cu chelating peptides. Sequential purification steps, including immobilised metal affinity chromatography (IMAC) and gel permeation chromatography (GPC) were employed for the separation of chelating peptides. GPC analysis showed that the mineral chelating peptides were mainly between 500 and 2000Da. Subsequent identification was carried out using UPLC–ESI-QTOF MS/MS. Overall, 10 sequences were identified as potential chelating peptides. The Ca, Fe and Cu chelating activity of GPAGPHGPPG was 11.52±2.23nmol/μmol, 1.71±0.17nmol/μmol and 0.43±0.02μmol/μmol, respectively. This study identifies collagen as a good source of peptides with potential applications as functional ingredients in the management of mineral deficiencies.

Abstract 5093: Nucleolin phosphorylation profiling under normal and cellular stress conditions

Abstract Nucleolin (NCL) is a ubiquitous nucleolar phosphoprotein that integrates critical cellular processes, from cell growth (ribosome biogenesis) and cell proliferation to cell cycle arrest and cell death. Deregulation of these processes often lead to different pathological conditions including cancer and heightened levels of nucleolin are often found in a variety of tumors. Nucleolin is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase 1 (Cdk1). Although increased nucleolin phosphorylation by CK2 is finely regulated in exponentially dividing cells, the role of nucleolin phosphorylation in regulating cell cycle remains largely unexplored. The high content of acidic stretches in the N-terminus of nucleolin protein limits the effective use of mass-spectrophotometry as well as our ability to generate various phosphorylation-specific nucleolin antibodies. In this study, we used novel phosphate-binding tag to analyze nucleolin phosphorylation in human osteosarcoma cells. With phosphate affinity gel electrophoresis and subsequent western analyses, we have enabled the detection and quantification of different phosphorylation states of nucleolin during interphase as well as in mitosis. CK2 phosphorylated nucleolin (as in interphase) has multiple bands that migrate slower when bound to phos-tag as compared to hypo-phosphorylated nucleolin mutant (6/S*A, where six consensus CK2 sites were modified from serine to alanine and hence defective in undergoing phosphorylation). In contrast, a single hyper-phosphorylated nuclolin band was predominant in mitotic arrested cells. Furthermore, we elucidated nucleolin phosphorylation patterns under various cellular stresses e.g. DNA damage (UV, CPT) as well as kinase stimulation (e.g. arachidonic acid) or inhibition by CK2-specific inhibitor CX4945. Our data provide for the first time informative nucleolin phosphorylation patterns during different cellular conditions. This profiling of nucleolin phosphoprotein is essential in understanding the role of nucleolin phosphorylation in various cellular processes as well as during cellular response to stress. This work is supported by grants PSC/CUNY 66740-00 44 and CA175794. Citation Format: Kenneth Ng, Allana Rodriguez, Anjana D. Saxena. Nucleolin phosphorylation profiling under normal and cellular stress conditions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2014-5093

Extracellular secretion of noncatalytic plant cell wall-binding proteins by the cellulolytic thermophile Caldicellulosiruptor bescii.

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (Ka) values of 5.2 × 10(6) to 44 × 10(6) M(-1) among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.

Preparation and antitumor effect of a toxin-linked conjugate targeting vascular endothelial growth factor receptor and urokinase plasminogen activator.

The aberrant signaling activation of vascular endothelial growth factor receptor (VEGFR) and urokinase plasminogen activator (uPA) is a common characteristic of many tumors, including lung cancer. Accordingly, VEGFR and uPA have emerged as attractive targets for tumor. KDR (Flk-1/VEGFR-2), a member of the VEGFR family, has been recognized as an important target for antiangiogenesis in tumor. In this study, a recombinant immunotoxin was produced to specifically target KDR-expressing tumor vascular endothelial cells and uPA-expressing tumor cells and mediate antitumor angiogenesis and antitumor effect. Based on its potent inhibitory effect on protein synthesis, Luffin-beta (Lβ) ribosome-inactivating protein was selected as part of a recombinant fusion protein, a single-chain variable fragment against KDR (KDRscFv)-uPA cleavage site (uPAcs)-Lβ-KDEL (named as KPLK). The KDRscFv-uPAcs-Lβ-KDEL (KPLK) contained a single-chain variable fragment (scFv) against KDR, uPAcs, Lβ, and the retention signal for endoplasmic reticulum proteins KDEL (Lys-Asp-Glu-Leu). The KPLK-expressing vector was expressed in Escherichia coli, and the KPLK protein was isolated with nickel affinity chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis test demonstrated KPLK was effectively expressed. Result of invitro cell viability assay on non-small cell lung cancer (NSCLC) H460 cell line (uPA-positive cell) revealed that KPLK significantly inhibited cell proliferation, induced apoptosis, and accumulated cells in S and G2/M phases, but the normal cell line (human submandibular gland cell) was unaffected. These effects were enhanced when uPA was added to digest KPLK to release Lβ. For invivo assay of KPLK, subcutaneous xenograft tumor model of nude mice were established with H460 cells. Growth of solid tumors was significantly inhibited in animals treated with KPLK up to 21 days, tumor weights were decreased, and the expression of angiogenesis marker CD31 was downregulated; meanwhile, the apoptosis-related protein casspase-3 was upregulated. These results suggested that the recombinant KPLK may have therapeutic applications on tumors, especially uPA-overexpressing ones.