重组小鼠IL-33成熟蛋白的原核表达、纯化及活性检测

目的为了在大肠埃希菌中表达具有生物学活性的小鼠IL-33成熟蛋白。方法利用PCR技术从pc DNA3.1-IL-33质粒中扩增小鼠IL-33成熟蛋白的基因,将其插入原核表达载体p ET21a(+),构建成p ET21a-m IL-33质粒,转化至大肠埃希菌BL21,筛选出可表达m IL-33的大肠埃希菌工程菌株。经IPTG诱导表达,用镍柱亲和层析法纯化。结果经SDS-PAGE分析获得纯度高达95%的m IL-33蛋白,相对分子质量18 000。ELISA试验显示m IL-33可以有效的促进肠系膜淋巴细胞产生Th2型细胞因子(IL-5),与未处理组的差异具有显著的统计学意义。结论利用大肠埃希菌表达系统表达了具有生物活性的m IL-33蛋白,为继续开展IL-33在炎症性肠病的免疫治疗研究奠定了基础。     

Production and purification of recombinant DP1B silk-like protein in plants

Spider dragline silk of Nephila clavipes consists of two highly repetitive proteins, spidroin 1 and spidroin 2. To develop a plant platform for production of recombinant silk-like protein, two plant-optimized DP1B genes were synthesized to mimic spidroin 1. DP1B-8P encodes for a 64 kD DP1B silk-like protein and DP1B-16P for a 127 kD DP1B silk-like protein. Both genes have been introduced into Arabidopsis for leaf-based production driven by the 35S promoter and for seed-specific production driven by the -conglycinin subunit promoter, respectively. They have also been expressed in somatic soybean embryos under the control of the -conglycinin subunit promoter. The results demonstrate the synthesis and accumulation of DP1B silk-like protein in leaves and seeds of Arabidopsis, as well as in somatic soybean embryos. They suggest that seeds are the more preferred tissue for DP1B production since they offer higher production yield and quality. In addition, a simplified protocol for purifying DP1B from plant tissue is discussed.     

Small-molecule catalysts of oxidative protein folding

Oxidative protein folding occurs both in vivo and in vitro and involves the formation and rearrangement of protein disulfide bonds (SS bonds). In vivo these reactions are catalyzed by enzymes, including the eukaryotic enzyme protein disulfide isomerase (PDI). Using the physical properties of PDI as a guide, several small-molecule catalysts of oxidative protein folding have been designed, synthesized, and tested. These small molecules can improve the folding rate of the model substrate ribonuclease A by a factor of over 10 and improve the yield by up to a factor of 3 over traditional conditions. The molecules have also been demonstrated to significantly improve the in vivo folding of proteins as well.     

Role of ascorbate in oxidative protein folding

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.     

Expression and purification of recombinant mouse fibrillarin.

Fibrillarin is a 34-kDa nucleolar protein associated with many of the small nucleolar ribonucleoprotein (snoRNP) particles and plays a role in ribosomal RNA processing. A subset of patients with the systemic autoimmune disease Scleroderma produce autoantibodies against fibrillarin and it is a genetically restricted target of murine mercury-induced autoimmunity. To aid in characterizing the antigenicity of fibrillarin, we have constructed two forms of mouse fibrillarin. The wild-type clone contains two cysteine residues that enable the protein to form an intramolecular disulfide bond, whereas the mutant clone contains alanine replacements which cannot form the disulfide bond. We have successfully expressed and purified both wild-type and mutant recombinant mouse fibrillarin using nickel-chelation chromatography. The combination of T7 promoter-driven expression vector pET28 and Escherichia coli strain JM109(DE3) induced at 25 degrees C yielded up to 19 mg of 94% pure recombinant protein per liter of culture. As the antigenicity of fibrillarin requires the full-length protein, the purification protocol was optimized for isolation of the full-length protein by the addition of N- and C-terminal T7 Tag and FLAG epitope sequences to the fibrillarin sequence. Anti-peptide antibodies were used in immunoblot to identify conditions favoring minimal proteolysis of recombinant protein. Both wild-type and mutant recombinant fibrillarin, purified under denaturing conditions and in the presence of 2-mercaptoethanol, were recognized by anti-fibrillarin antibodies from Scleroderma patients and exhibited structural similarities to eukaryotic and in vitro translated fibrillarin.     

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

艰难梭菌毒素A原核表达及重组蛋白纯化

目的构建艰难梭菌(Clostridium difficile,C.difficile)毒素A羧基末端原核表达载体,优化诱导表达条件及纯化重组蛋白。方法利用聚合酶链式反应扩增C.difficile毒素A羧基末端基因序列,并将此序列转入pET-28b载体,构建pET-28b-tcdA重组表达载体,并将表达载体转化到BL21(DE3)感受态大肠埃希菌细胞中,分别在不同条件下进行诱导表达,获得最佳诱导表达条件后进行大量表达,最后用Ni柱对重组蛋白进行亲和纯化,得到纯化后的重组蛋白。结果成功构建了pET-28b-tcdA重组表达载体,其重组蛋白表达最佳诱导条件:菌液吸光度值取0.6、温度取25℃、IPTG终浓度取1.0mmol/L、诱导时间取10h。蛋白纯化咪唑磷酸盐洗脱液最佳浓度取200mmol/L。结论成功构建pET-28b-tcdA重组表达载体,在大肠埃希菌BL21(DE3)中可以有效表达,并获得高浓度重组蛋白,为进一步制备TcdA适配子奠定了一定实验室基础。     

Human recombinant laminin-binding protein: isolation, purification, and crystallization

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.     

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni–NTA (Ni²⁺-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.     

Catalysis of oxidative protein folding by small-molecule diselenides

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low p K a of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.     

Production and purification of recombinant human oxytocin overexpressed as a hybrid protein in Escherichia coli

The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.     

Effective expression and purification of recombinant onconase, an antitumor protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.     

Production and purification of recombinant human glucagon overexpressed as intein fusion protein in Escherichia coli

Chemico-enzymatic synthesis and cloning in Esherichia coli of an artificial gene coding human glucagon was performed. Recombinant plasmid containing hybrid glucagons gene and intein Ssp dnaB from Synechocestis sp. was designed. Expression of the obtained hybrid gene in E. coli, properties of the formed hybrid protein, and conditions of its autocatalytic cleavage leading to glucagon formation were studied.     

Production and characterization of recombinant tachycitin,the Cys-rich chitin-binding protein

Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.     

Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin

The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.     

The oxidative protein folding machinery in plant cells

Formation of intra-molecular disulfides and concomitant oxidative protein folding is essential for stability and catalytic function of many soluble and membrane-bound proteins in the endomembrane system, the mitochondrial inter-membrane space and the thylakoid lumen. Disulfide generation from free cysteines in nascent polypeptide chains is generally a catalysed process for which distinct pathways exist in all compartments. A high degree of similarities between highly diverse eukaryotic and bacterial systems for generation of protein disulfides indicates functional conservation of key processes throughout evolution. However, while many aspects about molecular function of enzymatic systems promoting disulfide formation have been demonstrated for bacterial and non-plant eukaryotic organisms, it is now clear that the plant machinery for oxidative protein folding displays distinct details, suggesting that the different pathways have been adapted to plant-specific requirements in terms of compartmentation, molecular function and regulation. Here, we aim to evaluate biological diversity by comparing the plant systems for oxidative protein folding to the respective systems from non-plant eukaryotes.     

A first-order-like state transition for recombinant protein folding

Normally, proteins will aggregate and precipitate by direct folding processes. In this study, we report that quasi-static processes can restore both the structure and bio-function of two kinds of fish recombinant growth hormones (Plecoglossus altivelis and Epinephelus awoara). The conformational changes and the particle-size-distribution (PSD) of each refolding intermediate can be monitored by circular dichroism spectroscopy (CD) and dynamic light scattering (DLS), respectively. Conformation analysis of the CD spectra of the refolding intermediates indicated that the secondary structures were restored in the initial refolding intermediate. However, the tertiary interactions of the proteins were restored during the last two refolding stages, as elucidated by thermal stability tests. This is consistent with a sequential model. DLS analysis suggested that the average hydrodynamic radii of the refolding intermediates shrank to their native-like sizes after the first refolding stage. This is consistent with a collapse model. After comparison with the data on the direct folding process, it is concluded that the denaturant-containing protein folding reaction is a first-order-like state transition process.     

Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.