Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10⁵ Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K _d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K _d value of 10⁻⁷ M.     

Transgenic Expression and Functional Characterization of Human Platelet Derived Growth Factor BB (hPDGF-BB) in Tobacco (Nicotiana tabacum L.)

Recombinant human platelet derived growth factor BB (rhPDGF-BB) is clinically approved for treating diabetic neuropathic ulcers. Plant-based expression systems offer less expensive ways of producing recombinant drugs, which do not require purification for clinical use. From this perspective, rhPDGF-BB is an ideal candidate for expression in plants as it can be applied topically. Here, we report a proof of concept study, in which rhPDGF-BB was expressed in tobacco plants, and its biological activity was tested in vitro. The mature human platelet derived growth factor BB (hPDGF-BB) gene was codon-optimized for tobacco and fused with ER targeting and retention signals, 5′ and 3′ UTRs of arc5-1 gene along with CaMV 35S promoter, and then, transferred by Agrobacterium-mediated transformation. Gene and protein expression of hPDGF-BB were confirmed by PCR and immunoblot studies. Bioactivity of hPDGF-BB expressed protein was determined by in vitro assays such as proliferation and migration in NIH3T3 cells. Our data reveals that total soluble proteins containing hPDGF-BB from transgenic plants showed a 4.5-fold increase in fibroblast proliferation compared to non-transgenic plants. Furthermore, plant-made rhPDGF-BB induced chemotaxis of treated cells and promoted wound healing in vitro. These results clearly demonstrate that functionally active rhPDGF-BB protein can be produced in plants and might have therapeutic benefits.     

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration （ hALR）, Expression and Purification of hALR

Introduction Theliverisoneoftheorgansthathaspotentialre generativecapabilityinmammaliananimals[1].Studies oncaninemodelshaveindicatedthatthelivercanre generate,inonlytwoweeks,toitsoriginalsize,after70%hepatectomy[2].Therefore,theresearchoncellu larandmole…     

Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium

A key enzyme for the biosynthesis and bioengineering of heparin, 3-O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10⁴–10⁵-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.     

Enhanced In Vitro Refolding of Soluble Human Glucocorticoid-Induced TNF Receptor-Related Ligand

The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.     

Enteropeptidase

A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21.9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K _m -200μM and 125μM, respectively) were bound to the enzyme through the linker (Asp)₄ Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K _m=2.4μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding Avariation in the enzyme's uniquespecificity wasshown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466–800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118–465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.     

Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri

Background

Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host.

Results

In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.

Conclusions

Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.     

Aliphatic hydroxylation and epoxidation of capsaicin by cytochrome P450 CYP505X

Microbial cytochrome P450 enzymes (CYPs) are able to mimic the metabolism of human CYPs. One challenge is to identify the respective drug metabolites and to compare substrate specificities to those of the human enzymes. In this study, a class VIII self-sufficient CYP from Aspergillus fumigatus (CYP505X) and variants of this enzyme were heterologously expressed in E. coli. The substrate scope of the variants was determined using active pharmaceutical ingredients (APIs) and (hetero)cyclic compounds. Capsaicin – the active compound in chili peppers – was oxidized most efficiently (4.36?μM/min) in a whole cell mediated biotransformation. The products were isolated, purified and their structures elucidated by 1D and 2D NMR. The two major metabolites showed modifications on the lipophilic side chain. Specifically, capsaicin was hydroxylated at position 8 to give (E)-8-hydroxy-N-(4-hydroxy-3-methoxybenzyl)-8-methylnon-6-enamide and epoxidized at the double bond to give N-(4-hydroxy-3-methoxybenzyl)-5-(3-isopropyloxiran-2-yl)-pentanamide.     

Construction of Human Nonimmune Library and Selection of scFvs Against IL-33

Interleukin (IL) 33 plays very important roles in inflammatory and allergic diseases. To select human single-chain Fv fragments (scFvs) against IL-33, a nonimmune phage library system was constructed. The full-length cDNA library was synthesized for amplification of the variable heavy chain (V_H) and variable light chain (V_L). By overlapping extension PCR for splicing V_H and V_L, the full-length scFv library DNA were amplified and then transformed into Escherichia coli TG1. The scFv library was constructed successfully which contained 2.5?×?10⁸ independent clones with full-length scFv inserts. The results of fingerprint maps of the scFvs by BstN I and DNA sequencing from the library at random proved that the library was diverse. The human IL-33 was amplified, expressed, and purified. The purified IL-33 with bioactivity was biotinylated and used as antigen for selection of scFv library by phage display. After three rounds of affinity selection, about 30?% of clones have specific binding activity with IL-33. Five of those with good binding activity were transformed into E. coli strain HB2151 for soluble expression. The selected scFvs were further identified by western blot and sequencing. Those selected scFvs could be used for further research of their effect on inflammatory and allergic diseases such as asthma by blockade of IL-33.     

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.     

Completely dispersible PEGylated gold nanoparticles under physiological conditions: modification of gold nanoparticles with precisely controlled PEG-b-polyamine

A novel water-soluble, biocompatible polymer, poly(ethylene glycol)-block-poly((2-N,N-dimethylamino)ethyl methacrylate) (PEG-b-PAMA), possessing controlled molecular weight with a narrow molecular weight distribution, was synthesized by the atom-transfer radical polymerization (ATRP) method. PEG-b-PAMA having a short PAMA chain length was successfully synthesized under suitable polymerization conditions. Gold nanoparticles (GNPs) were modified using PEG-b-PAMA prepared under a variety of PEGylation conditions. Under alkaline conditions (pH >10) and an [N]/[GNP] ratio of more than 3300, the PEGylated GNPs (PEG-GNPs) showed complete dispersion stability, avoiding coagulation. The amino groups of the PAMA segment of the block copolymers were completely deprotonated above pH 10. This means that PEG-b-PAMA interacted with the GNP surface via multipoint coordination of the tertiary amino groups of PAMA, not electrostatically. The effect of the number of amino groups in the PAMA segment on GNP surface modifications was investigated by zeta potential and dynamic light scattering (DLS) measurements. When the PEG-GNPs were prepared in excess polymer solution, almost the same diameter was observed regardless of the PAMA chain length. After the PEG-GNPs were purified by centrifugation, the zeta potentials of all PEG-GNPs were shielded to almost 0 mV, indicating the effective modifications of the GNP surface by PEG-b-PAMA regardless of the chain length. However, the particle size and particle size distribution of the purified PEG-GNPs were strongly affected by the PAMA chain length. PEG-GNPs with longer PAMA segments underwent coagulation after purification, whereas PEG-GNPs with shorter PAMA segments increased their dispersion stability. The experimental results of the thermal gravimetric analysis confirmed that the PEG density on the GNP surface increased as the AMA units decreased to 3. Thus, the dispersion stability depended significantly on the PEG density on the GNP surface. GNPs modified with PEG-b-PAMA having short AMA units showed excellent dispersion stability under a variety of pH conditions. The excellent dispersion stability of the obtained PEG-GNP was also confirmed both in bovine serum albumin (BSA) solution and 95% human serum.     

不稳定性心绞痛血瘀证的血浆蛋白质组学研究

为了寻找冠心病不稳定性心绞痛血瘀证血浆差异表达蛋白, 探索冠心病不稳定性心绞痛血瘀证的蛋白质组学特点. 采用差异凝胶双向电泳和质谱联用技术对12例冠心病不稳定性心绞痛血瘀证患者和12例健康人血浆进行比较研究. 初步发现了Fibrinogen β chain, Fibrinogen γ chain, α1-Antitrypsin, Haptoglobin β chain, Haptoglobin α2 chain在冠心病不稳定性心绞痛血瘀证患者中高表达, ApoA-IV, ApoA-I, Transthyretin, ApoJ在冠心病不稳定性心绞痛血瘀证患者中低表达. 差异表达蛋白根据功能可分为以下三类: (1)急性时相反应负相蛋白; (2)载脂蛋白; (3)凝血相关蛋白. 冠心病不稳定性心绞痛血瘀证可能与炎症反应、脂代谢紊乱以及凝血功能异常相关.     

丝心蛋白基因分子克隆与表达的初步探讨

通过聚合酶链反应扩增丝心蛋白Ｃ亚基结构的基因，并将基克隆到融合蛋白表达载体ｐＲＩＴ２Ｔ质粒中得到ｐＲＩＴ２Ｔ－ＦＬ质粒，在大肠杆菌株Ｐ２３９２内进行表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和免疫印迹反应证明融合蛋白在大肠杆菌中得到了表达。     

Expression and Purification of ZNF191（243-368） in Three Expression Systems

ZNF191 （243-368）, a new human zinc finger protein, probably relates to some hereditary diseases and cancers, To obtain adequate amount of ZNF191（243-368） for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase （GST）, and hexahistidine （6 × His） were used and compared. Among these systems, the expression level of ZNFI91（243-368） was increased in inclusion body system under a higher isopropylthio-β-D-galactoside （IPTG） concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Use of a Bacterially Expressed Human Factor IX Light Chain to Develop Polyclonal Antibody Anti-hFIX

Hemophilia B is an X-linked recessive bleeding disorder caused by deficiency or malfunctioning of human coagulation factor IX (hFIX). Hemophilia B patients are treated at present by infusion of plasma derived hFIX which is not always efficient, because development of anti-hFIX antibodies (alloantibodies) in some cases inhibits the activity of the infused hFIX. The hFIX alloantibodies are directed against γ-carboxyglutamic acid residues (Gla-domain) or protease domain in hFIX light chain. An epitope-containing fragment of hFIX light-chain was expressed in a T7-based Escherichia coli expression system and after purification, it was used for the immunization of rabbit to develop specific antibodies anti-hFIX. The plasma, derived from the immunized rabbit, was shown to be able to detect the normal hFIX, which indicates for the presence of a specific anti-hFIX antibody and supporting that a bacterially expressed hFIX subfragment might be able to neutralize the alloantibodies. Considering the importance of hFIX and its related investigations, both the produced hFIX antigen and its corresponding antibody will play important roles for experiments dealing with the production of hFIX and studies involved in the neutralization of the hFIX inhibitors in hFIX-related disorders and other clinical applications.     

Undariase,a Direct-Acting Fibrin(ogen)olytic Enzyme from Undaria pinnatifida,Inhibits Thrombosis In Vivo and Exhibits In Vitro Thrombolytic Properties

A direct-acting fibrinolytic serine protease named undariase possessing anticoagulant and antiplatelet properties was purified from Undaria pinnatifida. Undariase showed a molecular weight of 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. It displayed a strong fibrin zymogram lysis band corresponding to the same molecular mass. The N-terminal sequence of undariase, LTATTCEELAAAPTD, does not match with any known fibrinolytic enzyme. The enzyme was stable and active at high temperatures (35–70 °C). The fibrinolytic activity of undariase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and 4-(amidinophenyl) methanesulfonyl fluoride (APMSF). The K _m and V _max values for substrate S-2251 were determined as 6.15 mM and 90.91 mM/min/ml, respectively. Undariase resulted in clot lysis by directly cleaving α and β chains of fibrin. Similarly, it preferentially acted on the Aα chain of fibrinogen followed by cleavage of the Bβ chain. It significantly prolonged the PFA-100 closure times of citrated whole human blood. In addition, undariase delayed the coagulation time and increased activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). Undariase exerted a significant protective effect against collagen plus epinephrine-induced pulmonary thromboembolism in mice. It prevented carrageenan-induced thrombus formation in the tail of mice. It also resulted in prolongation of APTT ex vivo. In conclusion, these results suggested a therapeutic potential of undariase for thrombosis.