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.     

Enhanced Expression and Primary Purification of Soluble HBD3 Fusion Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human β-defensin-3(HBD3) is a low molecular weight cationic peptide with a broad antimicrobial spectrum. A recombinant Escherichia coli (pET32-smHBD3) was constructed to produce HBD3 fusion protein (TrxA-HBD3) before, but the productivity is relatively low. In the present work, the effects of different expression conditions were systematically investigated to improve the expression level of the fusion protein. With regard to the volumetric productivity, the optimal conditions were determined as follows: cultivation at 34 °C in MBL medium, induction at middle stage of the exponential growth phase with 0.4 mM isopropylthio-d-galactoside, and postinduction expression for 8 h. Under these conditions, the volumetric productivity of the fusion protein reached 2.55 g/L, i.e., 0.55 g mature HBD3/L, which was about 2.6 times of that obtained under the unoptimized conditions. And the target protein still maintained high solubility (≥97.9%) and accounted for 66% of the total soluble protein. A cationic exchange purification step was employed to obtain high-purity target protein (90%) with a recovery ratio of 78%. This soluble expression level of HBD3 fusion protein was the highest among all the reported literature and facilitated the development of high efficient purification of HBD3.     

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.     

Purifying and Characterizing Bacterially Expressed Soluble Lactate Dehydrogenase from Plasmodium knowlesi for the Development of Anti-Malarial Drugs

Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.     

Recombinant Production and Characterization of SAC,the Core Domain of Par-4, by SUMO Fusion System

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137–195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.     

重组HEV衣壳蛋白截短体的表达及鉴定

戊型肝炎(HE)是由戊型肝炎病毒(HEV)感染引起的肠道病毒性传染病. HEV是一种无囊膜的单股正链RNA病毒, 其编码区由3个开放阅读框(ORF)组成, 属戊型肝炎病毒科. HEV衣壳蛋白由ORF2编码. 本研究根据编码HEV ORF2 aa382~aa674的核苷酸序列克隆了p293基因, 并将其克隆入原核表达载体pET28a, 利用大肠杆菌BL21(DE3)对HEV衣壳蛋白截短体(p293)进行了表达. SDS-PAGE和Western blot检测结果表明, 在优化的表达条件下(1 mmol/L IPTG, 250 r/min, 37℃, 5 h), 重组蛋白p293能够在大肠杆菌内有效表达, 目的蛋白约占总蛋白的66.15%. TEM检测结果显示, 原核表达的p293能够在体外形成约30~40 nm的病毒样颗粒. 免疫印迹和免疫荧光检测结果表明, p293与HEV标准阳性血清具有良好的反应原性和反应特异性. 实验结果表明, p293可应用于HEV宿主吸附和病毒装配研究, 为HEV的预防与诊断研究奠定了基础.     

Molecular cloning, expression and sequence analysis of DNA polymerase I from an Iranian thermophilic bacterium, Bacillus sp. G (2006)

Thermostable DNA polymerases are widely used in DNA amplification reactions such as the Polymerase Chain Reaction (PCR), requiring the activity of the enzymes at high temperatures. The aim of the present study was to assess the potential biotechnological capabilities of Iranian thermostable DNA polymerases. To this end, we cloned the gene encoding a DNA polymerase from a novel thermophilic eubacterium, Bacillus sp. G (2006). Phylogentic analysis of this gene revealed that the new isolate belongs to the genera Bacillus. Sequence analysis of the fragment produced by degenerate primers also showed that it consists of 2,631 bp encoding an 876 amino acid protein, and subsequent amino acid sequence analysis of this DNA polymerase showed that it belongs to family A-type DNA polymerases. The expression vector pET28a (+) was chosen for expression of the gene fragment in the mesophilic host bacterium E. coli BL21. This expression vector has some advantages such as attachment of a Poly-His tag to the N-terminus of the protein for the ease of purification and a powerful promoter of lac-Z induced by IPTG. The band corresponding to the protein product was observed in the molecular weight range of about 100KDa on the SDS-PAGE gel after heat and Ni⁺²-NTA column chromatography. Using the dot blot technique, the polymerase activity of the enzyme was qualitatively confirmed at 70 °C. Therefore, it is suggested that optimizations of this activity could make this enzyme appropriate for PCR processes in future.     

Visualizing Soluble Protein Mutants by Using Monomeric Red Fluorescent Protein as a Reporter for Directed Evolution

Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within the nadD gene, used to transform Escherichia coli DH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution.     

Maltose-binding Protein Improving the Crystallizability of C2 Domain of Human Coagulation Factor V

Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.     

Cloning and Expression of Che a 1, the Major Allergen of <Emphasis Type="Italic">Chenopodium album</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chenopodium album is a weedy annual plant in the genus Chenopodium. C. album pollen represents a predominant allergen source in Iran. The main C. album pollen allergens have been described as Che a 1, Che a 2, and Che a 3. The aim of this work was to clone the Che a 1 in Escherichia coli to establish a system for overproduction of the recombinant Che a 1 (rChe a 1). In order to clone this allergen, the pollens were subjected to RNA extraction. A full-length fragment encoding Che a 1 was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from extracted RNA. Cloning was carried out by inserting the cDNA into the pET21b (+) vector, thereafter the construct was transformed into E. coli Top10 cells and expression of the protein was induced by IPTG. The rChe a 1 was purified using histidine tag in recombinant protein by means of Ni–NTA affinity chromatography. IgE immunoblotting, ELISA, and inhibition ELISA were done to evaluate IgE binding of the purified protein. In conclusion, the cDNA for the major allergen of the C. album pollen, Che a 1, was successfully cloned and rChe a 1 was purified. Inhibition assays demonstrated allergic subjects sera reacted with rChe a 1 similar to natural Che a 1 in crude extract of C. album pollen. This study is the first report of using the E. coli as a prokaryotic system for Che a 1 cloning and production of rChe a 1.     

High Soluble Expression of <Emphasis Type="SmallCaps">d</Emphasis>-Amino Acid Oxidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Regulated by a Native Promoter

To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P_Hase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the P_Hase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m³ fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.     

人野生型MBL基因在大肠杆菌中的表达

为获得人MBL蛋白，并对其功能进行初步研究，用DNA重组法构建了组氨到标签融合原核表达质粒pET28(b)-MBL。将重组质粒转入大肠杆菌BL21（DE3），经IPTG在37℃条件下诱导培养，利用SDS－PAGE，Westem-blot检测目的蛋白的表达，用IMAC金属螯合层析柱对其进行纯化。成功地表达了重组MBL蛋白，纯化的MBL浓度约为844μg/mL，为制备MBL的基因工程抗体奠定了基础。     

Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5?°C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37?°C and in a buffer system of pH?6 to 7. The enzyme exhibited thermostability at 50?°C with a 33% and 28% of activity retention after 45 and 60?min. The kinetic parameters for the enzyme were calculated from Lineweaver?CBurk plot, and K _m and V _max were 0.89?mM and 0.18?U/mg, respectively.     

Cytokines Expression Induced by <Emphasis Type="Italic">Ganoderma sinensis</Emphasis> Fungal Immunomodulatory Proteins (FIP-gsi) in Mouse Spleen Cells

Ganoderma sinensis fungal immunomodulatory protein (FIP-gsi) was a new member of FIPs family. Based on the cloning of FIP-gsi gene from G. sinensis, this paper reported that FIP-gsi gene was expressed in Escherichia coli expression system. Then, the recombinant proteins were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Finally, the immunomodulatory activity was examined by inducing cytokine genes expression. The results showed that the recombinant FIP-gsi protein could be expressed in E. coli and got the yield of about 25% of the soluble form in the total soluble protein. The FIP-gsi protein was composed of 111 amino acids, and the sequence of homologous rate was 88.6% with FIP-glu (LZ-8). Furthermore, it could enhance the levels of interleukin (IL)-2, IL-3, IL-4, interferon gamma, tumor necrosis factor alpha, and IL-2 receptor (IL-2R) in mouse spleen cells.     

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.     

2,3-吡啶二甲酰亚胺及其工程菌转化产物的高效液相色谱检测

建立了利用含D-海因酶的基因工程菌转化吡啶二甲酰亚胺（PDI）生成3-氨基甲酰基-α-吡啶甲酸（α-3CP）的高效液相色谱（HPLC）检测方法。将工程菌pET3a-hyd/BL21（DE3）诱导表达后收集菌体,以PDI为底物,37℃摇床反应30 min后,以13000 r/min离心,取上清液进行HPLC检测。色谱条件：Hypersil^TM GOLD C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为H₂O-乙腈（体积比为90：10,含0.1%（体积分数）三氟乙酸）;检测波长为254 nm。当底物PDI达到饱和浓度时,测得工程菌pET3a-hyd/BL21（DE3）的比活力为0.61 U/（mL·10OD_{600 nm}）。该研究为今后利用生物法制备复杂半酰胺有机物提供了坚实的理论基础。     

High Levels of Expression of Fibroblast Growth Factor 21 in Transgenic Tobacco (<Emphasis Type="Italic">Nicotiana benthamiana)</Emphasis>

Fibroblast growth factor-21 (FGF21) is a hepatic hormone that plays a critical role in metabolism, stimulating fatty acid oxidation in the liver and glucose uptake in adipose tissue. In this study, we produced tobacco plants expressing human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the human FGF21 gene fused with green florescence protein and a histidine tag. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the hFGF21 gene was correctly translated in tobacco plants. Seven days after agroinfection, the recombinant hFGF21 had accumulated to levels as high as 450 μg g⁻¹ fresh weight in leaves of agroinfected plants. The recombinant hFGF21 was purified from plant tissues by Ni–NTA affinity chromatography, and the purified hFGF21 stimulated glucose uptake in 3T3/L1 cells. This indicated that the recombinant hFGF21 expressed via the PVX viral vector in N. benthamiana was biologically active.     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.