Cloning and expression of a novel antifreeze protein AFP72 from the beetle <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

A novel antifreeze protein AFP72 cDNA (GenBbank accession No. AY929389) was obtained by RT-PCR from Tenebrio molitor. The 216 bp fragment encodes a protein of 72 amino acid residues. Sequence analysis revealed that the cDNA displays a high degree of homology with T. molitor antifreeze proteins, ranging up to 90.78%. Recombinant plasmids pMAL-p2X-afp72 and pMAL-c2X-afp72 were transferred into E. coli TBI to induce a MBP fusion protein by IPTG. The target fusion protein was released from the periplasm and cytoplasm by the cold osmotic shock procedure and sonication respectively. The content of the fusion protein came up to 38.9 and 41.5% of the total dissolved protein, respectively. The fusion protein was purified through an amylose affinity column, and incised by factor Xa. Molecular sieve chromatography was used to achieve a high state of purity of the target protein. The purified target protein di splayed a single band in SDS-PAGE. The fusion protein was shown to increase resistance to low temperatures in bacteria. This finding could help in further investigations of the properties and function of antifreeze proteins.     

一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因,插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM,并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后,以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性,并利用肝素亲和层析纯化,得到电泳纯的融合蛋白。Western blot分析表明,该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示,融合蛋白具有促3T3Bal/b细胞分裂活性,其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白,可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽,抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明,回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。     

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.     

Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein

Recombinant human parathyroid hormone (hPTH)-(1-84) was obtained from Escherichia coli using a cleavable fusion protein strategy. The fusion protein contains residues 1-138 of human growth hormone as the amino-terminal region and residues 1-84 of hPTH as the carboxyl-terminal region. A 7-residue linker containing the recognition/cleavage sequence of the site-specific blood coagulation protease activated factor X (factor Xa) joins the two regions. Intact hPTH-(1-84) is released from this fusion protein by cleavage in vitro with factor Xa. The fusion protein was produced at a high level and formed inclusion bodies which allowed it to be easily purified by low speed centrifugation, with a yield of approximately 50 mg/liter of culture. After factor Xa cleavage and high performance liquid chromatography purification, highly purified hPTH was obtained, with a final yield of 1.5-3 mg/liter. Physical and biological characterization of the purified hormone demonstrated that it was intact and active hPTH-(1-84).     

乙型肝炎病毒表面抗原preS1在大肠杆菌中的表达与鉴定

本文报告利用pWR590质粒为载体,构建了含1ac启动子、β-半乳糖苷酶(1—590)基因、Xa因子的四肽识别位点和HBV preS1、preS2编码序列的表达质粒,并成功地在大肠杆菌中获得稳定表达。融合蛋白经Xa因子消化和高效液相层析,得到了preS1(1—91)纯肽。此肽特异性地与人肝细胞质膜结合,从而为肝细胞上存在preS1受体提供了直接的实验依据,也为分离和鉴定肝细胞上preS1受体打下了良好的基础。     

In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

We have developed a strategy for overproduction of spinach apocytochrome b6 as a fusion protein to maltose-binding protein (MBP) in Escherichia coli, using the expression vector pMal-c2. The fusion protein was purified to virtual homogeneity by gel filtration chromatography and the method of insertion of hemes into fusion protein was elaborated. The ambient and low-temperature absorption spectra of the reconstituted cytochrome b6 were similar to those of cytochrome b6 spectra in isolated proteins or cytochrome b6f complexes and are typical for bis-histidine ligated b-type cytochromes. Optical circular dichroism (CD) spectra of the visible region further confirmed the appropriate binding of hemes by the apocytochrome b6 protein. We found that the incorporation of hemes was required for the refolding of the cytochrome b6 protein into the more compact structure found in the native cytochrome protein. Heme staining experiments suggested that the two hemes in the reconstituted cytochrome b6 protein are bound with different affinities. The reconstituted cytochrome b6 protein was cleaved by Xa factor proteolysis from fusion protein and separated for characterization. The procedure presented in this work for reconstitution of hemes into the cytochrome b6 protein should provide an important tool for structure/function studies of membrane-bound cytochrome proteins.     

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.     

提高Xa因子酶切效率的策略

为提高Xa因子对融合蛋白CBD-IGF和CBD-PACAP的酶切效率 ,以便高效释放非融合的重组多肽 ,利用基因工程技术在两个融合蛋白中Xa因子识别位点 (Ile-Glu-Gly-Arg↓ )前均引入 7个氨基酸组成的富含甘氨酸柔性短肽 (Gly-Thr-Gly-Gly-Gly-Ser-Gly)。纤维素亲和层析纯化各个融合蛋白 ,比较Xa因子对引入短肽前、后融合蛋白的酶切效率。比较结果表明 :短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性 ;但总体上CBD-IGF对Xa因子的敏感性比CBD-PACAP低。此研究结果提供了一种提高Xa因子酶切效率的策略。     

Improved adsorption to starch of a beta-galactosidase fusion protein containing the starch-binding domain from Aspergillus glucoamylase.

We have previously shown (Chen et al., 1991) that a beta-galactosidase (beta-gal) fusion protein (BSB133) containing 133 amino acids (aa) from the C-terminus of Aspergillus glucoamylase (GA) adsorbs strongly to starch compared to beta-gal, due to the presence of the GA starch-binding domain. We have now made deletions at the N-terminus of this 133-aa region to test the minimal size required for starch binding of beta-gal fusion proteins. Three fusion proteins (BSB119, BSB103, and BSB80) were genetically engineered, containing 119, 103, and 80 C-terminal aa from GA, respectively. The fusion proteins were expressed in Escherichia coli and purified. Purified BSB119 adsorbed to native starch at least 2-fold more strongly than did BSB133 or fusion proteins with shorter tails. Adsorption isotherms generated over a wide range of initial concentrations indicated a 10-fold difference in the loading capacity of starch for BSB119 (36.5 mg of protein/g of starch) compared to beta-gal (3.7 mg of protein/g of starch). Adsorption constants calculated from the initial slopes of the isotherms indicated a nearly 30-fold difference in affinity to starch for BSB119 (Kad = 63 mL/g of starch) compared to beta-gal (Kad = 2.3 mL/g of starch). BSB119 in the presence of crude enzyme extracts also bound to starch with a high affinity compared to a beta-gal control. Potential applications of the starch-binding tail include enzyme immobilization to starch or recovery and purification of target proteins from crude extracts.     

Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.     

Expression and purification of a recombinant LL-37 from Escherichia coli

Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic alpha-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as (15)N, (13)C and/or (2)H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C(18) column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the (15)N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform (15)N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies.     

Purification and functional reconstitution of a truncated human Na(+)/glucose cotransporter (SGLT1) expressed in E. coli.

A truncated human Na(+)/glucose cotransporter (C(5), residues 407-664) was expressed and purified from Escherichia coli using a GST fusion vector and glutathione affinity chromatography. The truncated transporter (C(5)) was cleaved from GST-C(5) by Factor Xa proteolysis and purified by gel filtration chromatography. Up to 1 mg of purified GST-C(5) was obtained from 1 l bacterial culture. Reconstitution of both GST-C(5) and C(5) proteins into lipid vesicles resulted in 2.5-fold higher initial uptake rates of [(3)H]D-glucose into C(5)-proteoliposomes than into liposomes. Transport was stereospecific, saturable, and inhibited by phloretin. These properties are similar to those obtained for C(5) in Xenopus laevis oocytes, and provide additional evidence that the five C-terminal transmembrane helices in SGLT1 form the sugar translocation pathway.     

重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein.

Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.     

Characterization and modelling of the hydrophobic domain of a sunflower oleosin

The oleosins are a group of hydrophobic proteins present on the surface of oil bodies in seeds, where they are thought to prevent coalescence. They contain a central hydrophobic domain of 68-74 residues that is thought to form a loop into the triacylglycerol matrix of the oil body, but the conformation adopted by this sequence is uncertain. We have therefore expressed an oleosin cDNA from sunflower (Helianthus annuus L.) in Escherichia coli as a fusion with maltose-binding protein (MBP) and isolated a peptide corresponding to the hydrophobic domain by sequential digestion with factor Xa (to remove the MBP) followed by trypsin and Staphylococcus V8 protease to remove the N- and C-terminal domains of the oleosin. Circular dichroism spectroscopy of the peptide in two solvent systems chosen to mimic the environment within the oil body (trifluoroethanol and SDS) demonstrated high proportions of alpha-helical structure, with no beta-sheet. A model was therefore developed in which the domain forms an alpha-helical hairpin structure, the two helices being separated by a turn region. We consider that this model is consistent with our current knowledge of oleosin structure and properties.     

Production in Escherichia coli of porcine type-I collagenase as a fusion protein with beta-galactosidase.

Porcine type-I collagenase (Colg-1) was produced as a fusion protein in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to Colg-1. Recombinant collagenase (reColg-1) was biologically active in the form of a fusion protein and could be released by treatment with factor Xa to yield Colg-1 with the authentic N terminus (phenylalanine) found in vivo. The results show that reColg-1 produced in E. coli is folded correctly, cleaves type-I collagen into 1/4 and 3/4 fragments at the characteristic Colg-sensitive site, and is produced at high enough levels to generate a source of recombinant enzyme for x-ray crystallography studies.     

Daboxin P,a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A₂ activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca⁺² binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A₂ enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.     

Extracellular production system of heterologous peptide driven by a secretory protease inhibitor of Streptomyces

Summary The value of a heterologous peptide extracellular production system in Streptomyces using a secretory protease inhibitor, was examined. DNA was synthesized encoding apidaecin 1b (AP1), an interesting antibacterial peptide discovered in lymph fluid of the honeybee, and was joined to the Streptomyces subtilisin inhibitor (SSI) gene via a 12-bp nucleotide sequence corresponding to the amino acid sequence specific for cleavage by blood coagulation factor Xa. The fusion protein (SSI-AP1) could be expressed and excreted efficiently into the medium by culturing S. lividans 66 harbouring a plasmid vector constructed for SSI secretion, into which the synthetic DNA was introduced. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and amino acid analysis of the purified SSI-AP1 protided reasonable results of molecular size and composition value. Interestingly, SSI-AP1 protein showed bifunctional activity: inhibitory activity of SSI and antibacterial activity of AP1. The inhibitory activity against Escherichia coli could be also detected after the fusion protein was cleaved by factor Xa. The extracellular production system presented here should provide a useful tool for production, analysis of mode of action, and also for genetic improvement of antimicrobial peptides such as apidaecin.Offprint requests to: H. Momose     

Production of extracellular domain of human tissue factor using maltose-binding protein fusion system

Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.     

精子发生相关基因片段CG14的克隆及其蛋白的表达

为研究与精子发生相关的基因并探讨其功能 ,用差异显示法发现了 1个与精子发生相关的基因片段CG14 .将该基因片段克隆到表达载体pGEX 3X上 ,在大肠杆菌中表达了融合蛋白 .通过谷胱甘肽 Sepharose 4B亲和柱纯化该融合蛋白 .经Xa因子酶切后Western印迹方法证明 ,靶蛋白分子量约为 8kD ,与预期分子量相符 .用融合蛋白免疫家兔获得抗血清 .免疫印迹实验表明 ,血清中含有CG14蛋白的特异性抗体 ,为进一步研究CG14基因及其表达蛋白的功能打下基础