Rational design of combination enzyme therapy for celiac sprue

Celiac sprue (also known as celiac disease) is an inheritable, gluten-induced enteropathy of the upper small intestine with an estimated prevalence of 0.5%-1% in most parts of the world. The ubiquitous nature of food gluten, coupled with inadequate labeling regulations in most countries, constantly poses a threat of disease exacerbation and relapse for patients. Here, we demonstrate that a two-enzyme cocktail comprised of a glutamine-specific cysteine protease (EP-B2) that functions under gastric conditions and a PEP, which acts in concert with pancreatic proteases under duodenal conditions, is a particularly potent candidate for celiac sprue therapy. At a gluten:EP-B2:PEP weight ratio of 75:3:1, grocery store gluten is fully detoxified within 10 min of simulated duodenal conditions, as judged by chromatographic analysis, biopsy-derived T cell proliferation assays, and a commercial antigluten antibody test.     

Dihydroisoxazole analogs for labeling and visualization of catalytically active transglutaminase 2

We report the synthesis and preliminary characterization of "clickable" inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bioorthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay and could subsequently be used to visualize the active enzyme in?situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.     

A molecular warhead and its target: tissue transglutaminase and Celiac Sprue

The substitution of a glutamine residue with 6-diazo-5-oxo-norleucine (DON) transforms an immunodominant gluten peptide into a potent inhibitor of tissue transglutaminase. DON-modified peptides could be useful for the study and therapy of celiac sprue.     

Structure, function and biological relevance of prolyl oligopeptidase

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze proteins and peptides containing more than 30 residues. The crystal structure of prolyl oligopeptidase (POP) has shown that the enzyme is composed of a peptidase domain with an alpha/beta hydrolase fold and a seven-bladed beta-propeller domain. This domain covers the catalytic triad and excludes large, structured peptides from the active site. The mechanism of substrate selection has been reviewed, along with the binding mode of the substrate and the catalytic mechanism, which differ from that of the classical serine peptidases in several features. POP is essentially a cytosolic enzyme and has been shown to be involved in a number of biological processes, but its precise function is still unknown. Many reports addressed experimentally the possible role of POP in cognitive and psychiatric processes, its involvement in the inositol phosphate signaling pathway, and its ability to metabolize bioactive peptides. Inhibitors were designed to reveal the cellular functions of POP and to treat neurological disorders. Other studies concerned the cellular localization of POP, its presumed interaction with the cytoskeletal elements, and its involvement in peptide/protein transport/secretion processes. The possible role of POP in Alzheimer disease is an intriguing issue, which is still debated. Recently, recombinant bacterial POPs have been investigated as potential therapeutics for celiac sprue, an autoimmune disease of small intestine caused by the intake of gluten proteins.     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

Solid-phase synthesis of a farnesylated CaaX peptide library: inhibitors of the Ras CaaX endoprotease

A solid-phase method, based on Kaiser's p-benzophenone oxime resin, was developed for the synthesis of a series of N-acetyl-S-(E, E-farnesylated) Ca(1)a(2)X tetrapeptides as potential inhibitors of recombinant Ras and a-factor converting enzyme (RCE). N-Acetyl-S-(E, E-farnesyl)-L-cysteine was coupled to resin-bound a(1)a(2) dipeptide using HOBt/DCC activation in conjunction with N-BOC chemistry. The protected farnesylated tripeptide was cleaved from the resin with simultaneous addition of the X residue by treating the resin-bound farnesylated Ca(1)a(2) tripeptide with L-amino acid benzyl ester tosylates under mildly acidic conditions. The benzyl ester was saponified, and the resulting carboxylate precipitated by ether to afford a library of tetrapeptides as a mixture of diastereomers at the cysteine center. The peptides were evaluated as inhibitors of recombinant yeast RCE endoprotease (yRCE) to obtain information about the affinity of the enzyme for the a(1)a(2)X portion of the Ca(1)a(2)X moiety.     

Basic studies of hydrogen evolution by escherichia coli containing a cloned citrobacter freundii hydrogenase gene

Citrobacter freundii genes that complemented Escherichia coli hyd-(hydrogenase activity) mutation were cloned in plasmids pCBH4 (6.2 kb) and pCBH6(5.7 kb). Hydrogen evolution by the transformant E. coli HK-8(pCBH4 or pCBH6) was investigated. The optimum culture temperature of recombinant E. coli cells for hydrogen evolution from glucose was in the neighborhood of 18 degrees C. The recombinant E. coli cells cultured at this condition showed a several-fold increase of hydrogen evolution, as compared with that of the wild-type cells. The plasmid-retention stability of this recombinant E. coli was extremely high, especially plasmid pCBH4, which was completely retained during 2 wk without any restriction. Hydrogen production by immobilized recombinant E. coli was then investigated using cells cultured at 18 degrees C. The hydrogen evolution rate from glucose and Lennox-broth were about twofold higher than that of E. coli C600, and this high hydrogen evolution rate was maintained for more than 1 mo.     

Linoleic Acid Isomerase from <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>: Purification,Characterization, Molecular Cloning,and Heterologous Expression

Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors.     

cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

Introduction Theleukemiainhibitoryfactor(LIF)isamulti functionalcytokinebelongingtotheinterleukin6(IL6)family[1].Itwasfirstfoundasamyeloidleukemic cellproliferationsuppressoranddifferentiationinducer cytokine[2].ThenaturalformofLIFisa38to67kDa glycosylate…     

来源于类芽孢杆菌属新型耐有机溶剂脂肪酶的克隆、表达与性质研究（英文）

脂肪酶(EC 3.1.1.3)全称为三酰基甘油水解酶,是一类能够将长链脂肪酸甘油酯水解成脂肪酸和二甘酯、单甘酯或甘油的酯键水解酶.它除了能够水解脂肪外,还具有催化酯化反应、酯交换反应、酸解反应、醇解反应以及氨解等反应的性质.在脂肪酶催化的反应中,通常用有机溶剂代替水.有机溶剂可以转移合成反应的平衡方向,通过溶剂工程修饰酶的选择性能够提高底物的溶解度、有机相产物的回收率、酶的热稳定性.但有机溶剂对酶活性和稳定性有不同程度的影响.因此,寻找在有机溶剂中表现出高活性和稳定性的脂肪酶是一个亟待解决的重要课题.由于微生物种类多、作用底物专一性强,且微生物来源的脂肪酶一般分泌到胞外,因此微生物脂肪酶是工业用脂肪酶的重要来源.目前,微生物脂肪酶的研究主要集中于根霉属(Rhizopus)、曲霉属(Aspergillus)、青霉属(Penicillium)、毛霉属(Mucor)、地霉属(Geotrichum)、假丝酵母属(Candida)、假单胞菌属(Pseudomonas)、伯克霍尔德菌属(Burkholderia)等具有工业应用价值的菌株.很少有类芽孢杆菌属所产脂肪酶进行相关酶学性质的研究.我们以Paenibacillus pasadenensis CS0611为出发菌株,在全基因序列草图中得到了一个新型脂肪酶基因lp2252.以Paenibacillus pasadenensis CS0611基因组为模板,设计特异性引物对目标序列进行扩增,并成功将其插入到表达载体p ET-28a中得到含有目的基因的重组质粒.在E.coli BL21(DE3)中,脂肪酶lp2252经0.1mmol/L的IPTG诱导后在20°C实现了高水平表达.重组脂肪酶的活性约为野生型的1631倍.用镍离子亲和层析柱快速、高效地纯化了两端带有组氨酸标签的重组脂肪酶,回收率为63.5%,纯化因子为10.78.纯化后的脂肪酶最适温度为50°C,在20-40°C范围内具有良好的稳定性.最适pH值为7,属于中性脂肪酶,同时在pH 3.0-8.0间具有较高稳定性.在金属离子如钙、镁离子和一些非离子表面活性剂的作用下,其活性有所提高.此外,纯化后的脂肪酶可被一系列水溶性有机溶剂激活,例如一些短链醇.而对某些水不溶性有机溶剂,其也具有高度的耐受性.综上所述,本文所涉新型脂肪酶在非水相催化领域具有广泛的应用和前景.     

产甘油基因工程菌的构建

利用途径工程的基本原理,在大肠杆菌中构建一条产甘油的新代谢途径。从酿酒酵母Sacchdromyces cerevisiae INVSc1菌株总DNA克隆3-磷酸甘油脱氢酶基因(gpdl)和3-磷酸甘油酯酶基因(hot2),构建由两个杂合启动子trc启动基因的双表达盒的重组质粒pGEM-Cgpd1-Chor2,后者转入E．coli JM109菌株,构建的重组菌株就具有一条直接将葡萄糖转化为甘油的新代谢途径,将该重组菌株以葡萄糖为底物进行摇瓶发酵,甘油产率为1．18g／L。该研究结果为进一步构建生产1,3-丙二醇工程菌打下了基础。     

Two enzymes catalyze the maturation of a lasso peptide in Escherichia coli

Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.     

具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni²⁺的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.     

Ligation independent cloning (LIC) as a rapid route to families of recombinant biocatalysts from sequenced prokaryotic genomes

A technique is presented for the high throughput generation of families of recombinant biocatalysts sourced from prokaryotic genomes, providing rapid access to the naturally evolved diversity of enzyme specificity for biocatalyst discovery. The method exploits a novel ligation independent cloning strategy, based on the locally engineered vector pET-YSBLIC and has been used for the rapid generation of a suite of expression plasmids containing genes encoding a family of six Baeyer-Villiger monooxygenases (BVMOs) from Mycobacterium tuberculosis H37Rv (MTb). The six resultant recombinant strains of E. coli B834 (DE3) expressing the genes were assayed for oxygenating activity in respect of the target reaction; the resolution of bicyclo[3.2.0]hept-2-en-6-one. The analysis of biotransformations catalysed by growing cells of E. coli was complicated by the production of indole in the reaction mixtures, possibly resulting from the in vivo activity of E. coli tryptophanase. Four of the recombinant strains expressing different BVMOs catalysed the oxidation of one or more of four screening substrates, well above controls that had been transformed with the re-ligated parent vector. One of the recombinant strains, E. coli B834 (DE3) pDB5, expressing the Rv3049c gene from MTb, was found to effectively resolve the target substrate, yielding a 19% yield of (1R, 5S)-(+)-bicyclo[3.2.0]hept-2-en-6-one with >95% enantiomeric excess in a 4 L fermentation reaction.     

A Novel Method for Conversion of Valuable BDS Intermediates HPBS／Cx-HPBS from DBT／Cx-DBT

A simple recombinant PCR method was used to delete the dszB gene responsible for the slowest step of the Dsz pathway and allow the accumulation of hydroxyphenyl benzene sulfinate (HPBS) in the recombinant E. coll. Using GC/MS, HPBS accumulation was confirmed. The recombinant E.coli could also desulfurize Cx-DBT to corresponding Cx-HPBS. The result gave a new insight to BDS process and explored a new method to obtain valuable surfactants from cheap raw materials.     

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.     

Streptomyces maritimus苯丙氨酸变位酶的制备、表征及催化合成β-芳香丙氨酸

克隆Streptomyces maritimus来源的苯丙氨酸变位酶(Sm PAM)基因,并进行异源表达,制备了重组Sm PAM,用于一步合成高附加值的β-芳香丙氨酸.提取S.maritimus的基因组,设计1对特异性引物,采用PCR扩增编码Sm PAM的结构基因pam,与表达载体p ET28a连接,构建重组表达质粒p ET28a-pam,转入E.coli BL21中表达,采用亲和层析柱分离纯化重组酶Sm PAM.结果表明,克隆得到编码523个氨基酸长度的Sm PAM基因pam,并在大肠杆菌中实现了高效表达,制得电泳纯的重组Sm PAM.该酶在最适温度30℃,p H=9.0条件下活力达到2.5 U/mg,具有较高的热稳定性和p H稳定性,在60~70℃下保持3 h未见活性下降,在p H=9~11保持24 h,残余酶活力达到98%.Sm PAM具有较宽的底物谱,可催化苯环上携带不同基团的3-芳香丙烯酸合成β-芳香丙氨酸,当苯环上携带吸电子基团时催化反应更易完成,其中2-硝基-β-苯丙氨酸的产率最高,达到93%.     

新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白GRFT基因氨基酸序列,推测其DNA编码序列,密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a（+）中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白. SDS-PAGE、Western Blot分析结果表明,目的蛋白得到良好表达并具有抗原活性;对表达条件进行优化,在最佳表达条件下,目的蛋白的表达量可占菌体总蛋白的55.84%;利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化,凝胶成像系统扫描分析表明,纯度可达94.06%;运用Dot-ELISA法进行复性蛋白的抗原结合活性检测,结果显示,目的蛋白能够与HIV-1膜蛋白抗原特异性结合,初步证明所表达的重组蛋白具有良好的体外结合活性;基于制备的能够表达HIV-1 gp120基因的靶细胞模型,运用IFA法开展目的蛋白的细胞结合活性研究,结果显示,目的蛋白能够与靶细胞发生特异性反应,表明成功制备并获得了具有生物活性的新型抗HIV-1蛋白GRFT,为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了坚实基础.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.