Cloning and expression of the haloalkane dehalogenase gene dhmA from Mycobacterium avium N85 and preliminary characterization of DhmA

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.     

The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution

Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.     

Biodegradation of 1,2,3-Trichloropropane through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.     

Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究

目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40～45℃,最适pH为7.0～7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

Cloning and Tissue Expression Characterization of the Chicken APOB Gene

Apolipoprotein B (APOB) serves an essential role in the assembly and secretion of triglyceride-rich lipoproteins and lipids transport. This study was designed to clone the full-length cDNA of the chicken APOB gene, to characterize the expression profile, and investigate the differential expression between layer and broiler of the chicken APOB gene. The full-length cDNA sequence (14,150-bp) that contained a 13,896-bp ORF encoding 4,631 amino acids was obtained by RT-PCR, RACE, and bioinformatics analysis. qReal-Time PCR analysis showed that the chicken APOB gene was highly expressed in kidney, liver, and intestine. The results of differential expression showed that the APOB gene was more highly expressed in intestine and kidney in Bai'er layer than in broiler, but there was no significant difference in liver between the two breeds. The results of this study provided basic molecular information for studying the role of APOB in the energy transportation in avian species.     

结核杆菌eis基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建结核分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物活性。方法:采用PCR技术克隆结核分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV-eis,经酶切和测序鉴定其正确性,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中,采用SDS-PAGE和Western blot检测eis基因在耻垢分枝杆菌中的表达。结果:成功构建结核杆菌eis基因穿梭表达载体pMV-eis;生长曲线说明重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE 和Western blot检测证实eis在耻垢分枝杆菌中可表达出相对分子量约42kDa的Eis蛋白。结论:成功构建了eis基因穿梭表达质粒pMV-eis,且该重组质粒在耻垢分枝杆菌中具有生物活性,为下一步研究表达产物Eis的功能奠定了一定基础。     

诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达

2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。     

Characterization and expression of secA in Mycobacterium avium

Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water). Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host. Using a combination of cloning and information available in the databank, we characterized the secA gene from M. avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics. In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp. It was determined that the secA of M. avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae. secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase. secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation. Future evaluation of the sec pathway in M. avium might provide important information about secreted proteins that are required for survival in different environments.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

结核分枝杆菌Rv0859基因的克隆、表达、纯化及蛋白的亚细胞定位

本研究体外克隆了结核分枝杆菌Rv0859基因, 融合表达并纯化了Rv0859蛋白。首先提取H37Rv标准菌株中的基因组DNA, 设计Rv0859基因两端的引物, 以H37Rv基因组DNA为模板通过PCR方法扩增Rv0859基因。用Hind III和BamHⅠ两种限制性内切酶双切Rv0859基因, T4连接酶连接到pET30载体上, 再转入大肠杆菌JF1125中, 经过筛选鉴定后抽提质粒测序, 得到重组正确载体, 转化到表达宿主大肠杆菌BL21中。用IPTG进行诱导表达, 通过聚丙酰胺凝胶电泳(SDS-PAGE)及质谱鉴定重组表达蛋白。0.05 mol/L浓度的IPTG 37°C诱导4 h重组蛋白的表达量最高。制备重组蛋白的多克隆抗体, 通过亚细胞分离及Western-blotting分析蛋白的亚细胞定位。结果成功地构建原核表达载体pET30a-Rv0859, 并获得47845 D左右的大量表达的Rv0859蛋白, Western-blotting结果表明Rv0859蛋白主要定位于细胞膜中, 微量存在于细胞壁中, 为进一步的Rv0859蛋白功能研究奠定了一定的基础。     

结核分枝杆菌Rv1884c基因的克隆及其原核表达

目的:构建结核分枝杆菌Rv1884c基因的原核表达质粒,获得结核分枝杆菌Rv1884c基因的表达蛋白。方法:制备结核分枝杆菌基因组DNA,采用聚合酶链反应技术扩增目的基因片段;通过pGEX-4T-1构建质粒载体pGEX-4T-1-Rv1884c,经序列测定证实正确后转化大肠杆菌DH5α,再经IPTG诱导表达GST-1884融合蛋白;用聚丙烯酰胺凝胶电泳分析重组蛋白的相对分子质量及表达形式。结果:扩增出了结核分枝杆菌Rv1884c基因,构建了具有正确基因序列的质粒载体pGEX-4T-1-Rv1884c,转化大肠杆菌DH5α后经诱导产生了高水平的表达产物。结论:构建了pGEX-4T-1-Rv1884c质粒载体,并诱导表达了GST-1884融合蛋白,为进一步研究Rv1884c蛋白的活性及其功能,探讨结核分枝杆菌快速促生长作用奠定了基础。     

小鼠巢蛋白基因结构及其转录调控元件的初步鉴定

通过筛选小鼠基因组ＢＡＣ文库,得到小鼠巢蛋白基因组２３．３ｋｂ ＤＮＡ序列,其中约１５．３ｋｂ已完成测序。与小鼠ｃＤＮＡ序列比较结果表明,小鼠巢蛋白基因包含３个内含子。与大鼠及人巢蛋白基因相比,小鼠巢蛋白基因中３个内含子的位置及大小均很保守。利用神经发育的体外实验模型,检测小鼠巢蛋白基因第２个内含子对荧光素酶报告基因的调控活性。系列缺失质粒转染细胞显示,小鼠巢蛋白基因第２个内含子对报告基因的转录具     

Cloning and Expression of the Inositol Monophosphatase Gene from Methanococcus jannaschii and Characterization of the Enzyme

Inositol monophosphatase (EC 3.1.3.25) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (K_m = 0.091 ± 0.016 mM; V_max = 9.3 ± 0.45 μmol of P_i min⁻¹ mg of protein⁻¹) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg²⁺ requirement, Li⁺ inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li⁺ and high concentrations of Mg²⁺ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.The sole pathway for myo-inositol biosynthesis is the cyclization of glucose-6-phosphate to inositol-1-phosphate (I-1-P) by I-1-P synthase (EC 5.5.1.4) and the dephosphorylation of I-1-P by inositol monophosphatase (I-1-Pase; EC 3.1.3.25) (7–9, 12, 16, 24). This de novo pathway provides the ultimate source of free inositol for the cell. It is also a key enzyme involved in second-message signal transduction processes in mammalian and plant cells (2, 24, 28, 37). In phosphoinositide signaling (2, 37), I-1-Pase recycles the water-soluble phospholipase C phospholipid degradation products, inositol phosphates, to myo-inositol and helps to maintain a moderate inositol pool. Its inhibition by millimolar concentrations of lithium (19) has made it a putative target of lithium therapy for manic depression (34).Di-myo-inositol-1,1′-phosphate (DIP), a novel inositol phosphate compound found in hyperthermophilic archaea, including Pyrococcus woesei (43), Pyrococcus furiosus (41), Methanococcus igneus (11), and Thermotoga maritima (36), is used for osmotic balance at high growth temperatures. In order to understand what regulates its accumulation in cells, the DIP biosynthetic pathway must be well characterized in vitro. Based on ¹³C-labeling studies and assays of crude protein extracts from M. igneus (10), a pathway was proposed that converts glucose-6-phosphate to I-1-P (step 1), hydrolyzes some of the I-1-P to myo-inositol (step 2), and activates I-1-P to CDP-inositol (CDP-I) (step 3) for a final reaction (step 4) whereby CDP-I is coupled to myo-inositol, generating DIP and CMP (Fig. ​(Fig.1).1). Activities for I-1-P synthase, I-1-Pase, and DIP synthase in the DIP biosynthetic pathway have been detected in crude protein extracts of M. igneus (10). Phosphatase activities are ubiquitous in cells, and the observed activity in M. igneus could be due to a specific I-1-Pase activity or a nonspecific phosphatase. For mammalian and plant cells, I-1-Pases are all lithium sensitive and are inhibited at millimolar concentrations of Li⁺ (14, 15, 19, 30, 42). The partially purified phosphatase in M. igneus exhibited substrate specificity for dl-I-1-P over other sugar phosphates (10). It had an absolute requirement for Mg²⁺, a characteristic of all specific I-1-Pases studied thus far, and was also partially inhibited by Li⁺, though at a much higher concentration (160 mM for 50% activity inhibition) than reported for I-1-Pases from other cells. While this was suggestive of a specific I-1-Pase, the same protein fractions demonstrated considerable activity toward p-nitrophenylphosphate (pNPP), a very poor substrate for mammalian enzymes (1, 14). These preliminary characterizations of phosphatase activity suggested that archaeal I-1-Pases might be different from mammalian and plant enzymes. Open in a separate windowFIG. 1Proposed DIP biosynthetic pathway. Glucose-6-phosphate is converted to I-1-P (step 1), some of which is hydrolyzed to myo-inositol (step 2), and I-1-P is activated to CDP-I (step 3) for a final reaction in which CDP-I is coupled to myo-inositol (step 4), generating DIP and CMP.Methanococcus jannaschii was the first archaeon whose complete genomic sequence was determined (6). Of all the archaea with sequenced genomes, it is the closest to M. igneus. MJ109 encodes a 252-amino-acid protein that is highly homologous to both I-1-Pase and extragenic suppressor (the suhB gene product) (6). The latter gene product cloned in E. coli also has I-1-Pase activity (29). The putative identification of the MJ109 gene product as an I-1-Pase prompted us to express the gene product in E. coli and to examine its specific activity toward a variety of phosphate esters. The protein produced in this fashion clearly has I-1-Pase activity and shows several striking differences from plant and mammalian I-1-Pase activities.     

Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205

The enzymes LinB_UT and LinB_MI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB_MI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB_MI were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB_MI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB_MI. The dynamics simulation analyses of wild-type LinB_MI and LinB_UT revealed that the entrance of the substrate access tunnel of LinB_UT was more flexible than that of LinB_MI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.