兔肝细胞色素b5在大肠杆菌中的表达

利用体外特定点突变技术,将原核生物核糖体结合位点及十碱基间隔子(spacer)引科兔肝经胞色素b_5 cDNA的5′端,并构建了一个兔红血球b_5cDNA克隆,重组入表达载体pKK223-3质粒中,在大肠杆菌中成功地表达了兔肝和红血球两种形式的b_5蛋白。产量分别为每毫升培养液1.5μg和4.5μg。     

PCR依赖型方法构建高质量酵母基因突变文库

针对定向进化中利用亚克隆的方法建立酵母突变文库建库周期长、效率低、库容量低、丰度低等问题,建立了一种基于体内同源重组构建酵母整合型基因突变文库的新方法。步骤为：构建目标基因的重组表达质粒;以此为模版,设计长引物片段,PCR/易错PCR/DNA Shuffling等方法扩增得到两端带有与表达载体40~70 bp同源序列的突变基因;再利用PCR扩增得到表达载体;将扩增得到的目标基因和表达载体以一定的摩尔比混合电转化酵母,目标基因和表达载体在酵母体内同源重组成为完整的表达盒,整合入酵母基因组,获得基因突变文库。对构建的突变文库进行筛选,分别得到了酶活、蛋白表达量及热稳定性提高的突变株。该方法为完全PCR依赖型 (PDM),在体内构建表达盒,效率高,操作方便,将建库周期由2周缩短为3 d,将库容量从传统的103~104提高到105以上,库阳性率达到95％。     

Temporal and spatial gene expression of cytochrome B5 during flower and fruit development in olives

We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4–6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 °C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.     

Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary cis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5' long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Psi) is located within an area which includes the 5' LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.     

北柴胡细胞色素P450酶基因的克隆及其植物过量表达载体的构建

目的:克隆北柴胡中可能参与柴胡皂苷生物合成的细胞色素P450酶基因,构建其过量表达载体,为通过转基因验证其功能奠定基础。方法:在454高通量测序获得5'和3'端部分cDNA序列的基础上,利用LD-PCR方法获得全长cDNA,根据全长cDNA序列设计含有酶切位点的PCR引物,利用高保真酶,以RNA反转录产物为模板PCR扩增细胞色素P450酶基因的开放读框,扩增产物与pEASY-T1 Simple载体连接,转化大肠杆菌DH5α;重组质粒pT1-P450经菌液PCR和酶切方法验证后测序,采用NCBI在线Blastx、DNAman和MEGA4软件对序列进行生物信息学分析,随后将pT1-P450的酶切产物插入双元载体pCAMBIA-SUPER 1300,菌液PCR和酶切验证重组质粒p1300-P450。结果:扩增到了北柴胡细胞色素P450酶基因BcCYP87E,构建了这一基因的过量表达载体。结论:细胞色素P450酶基因的克隆和转基因载体的构建,为后续开展转基因研究,验证其生物功能奠定了基础。     

Identification of Reo1, a Gene Involved in Negative Regulation of Cox5b and Anb1 in Aerobically Grown Saccharomyces Cerevisiae

In Saccharomyces cerevisiae, the COX5a and COX5b genes constitute a small gene family that encodes two forms of cytochrome c oxidase subunit V, Va and Vb, either of which can provide a function essential for cytochrome c oxidase activity and respiration. In aerobically grown wild-type yeast cells, Va is the predominant form of subunit V. The COX5b gene alone does not produce enough Vb to support a respiration rate sufficient to allow growth on nonfermentable carbon sources. By selecting for mutations that increase the respiratory capacity of a strain deleted for COX5a, we have identified a gene that is involved in negative regulation of COX5b expression under aerobic growth conditions. Each of four independently isolated reo1 mutations are shown to be recessive, unlinked to COX5b, but dependent on COX5b for phenotypic expression. The mutations define a single complementation and linkage group: designated as REO1 for regulator of expression of oxidase. reo1 mutations increase expression of COX5b in aerobically grown cells, but not in anaerobically grown cells, where expression is already elevated. These mutations have no effect on COX5a, the other member of this small gene family which is positively regulated by heme and oxygen. The REO1 gene does play a role in repression of ANB1, a gene that is normally repressed under aerobic but not anaerobic conditions. Neither rox1 or rox3 mutations, which have previously been shown to increase ANB1 expression, are in the same complementation group as reo1 mutations.     

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.     

Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5)

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b5 as a GST-cyt b5 fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein.     

A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C

Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35 degrees C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35 degrees C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 alpha subcomplex 4, cytochrome b5-like protein, apolipoprotein A-V, cathepsin D, pancreatic Rnase, and HMG-1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4 degrees C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems.     

Screening and purification for novel cytochrome b5 from uncultured environmental micro-organisms

AIMS: We describe a sequence-based PCR method suitable for the isolation of a novel soluble heme-binding domain of cytochrome b(5) (cyt b(5)) gene directly from metagenomic DNA is described. METHODS AND RESULTS: Using the degenerate primer set, a cyt b(5) gene was isolated directly from metagenomic DNA. Based on the sequence-based PCR method, the similar conserved motif of cyt b(5) from Rhodopseudomonas palustris strain makes the novel target gene. The gene encoding cyt b(5) was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized. CONCLUSIONS: Sequence-based strategy is an effective method for application of the novel gene from metagenomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of novel genes from metagenome, most of the micro-organism species are largely untapped, could represent an interesting and useful reservoir for biological processes.     

Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells

Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.     

Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.     

Splicing factor SF3b as a target of the antitumor natural product pladienolide

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.     

Site-specific integration of a transgene mediated by a hybrid adenovirus/adeno-associated virus vector using the Cre/loxP-expression-switching system

As vectors, adenoviruses (Ads) have many attractive advantages for in vivo gene therapy. However, Ads do not usually integrate into the host genome and gene expression is, thus, transient. Adeno-associated virus (AAV) integrates into a specific locus (AAVS1) on the human host's chromosome 19, while conventional recombinant AAV (rAAV) vectors do not possess this property because such vectors lack the rep gene. AAV vectors carrying the rep gene do not have enough space for insertion of a transgene. We have constructed a hybrid adenovirus/adeno-associated virus (Ad/AAV) vector which has the advantages of both Ads and AAVs. Given that the rep gene products inhibit propagation of Ads, we used the Cre/loxP-expression-switching system to regulate the expression of the rep gene. The Ad/AAV vector easily propagates, can efficiently infect a broad range of cell types, and can integrate into a specific locus on host chromosomes.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.