双质粒系统共表达大肠杆菌肉碱脱水酶基因caiB及其辅酶合成基因caiE

caiB基因和caiE基因分别编码肉碱脱水酶及其辅因子合成酶 ,两者的共表达可以获得高活性肉碱脱水酶的重组菌。分别用两相容性质粒和不相容质粒共表达肉碱脱水酶及其辅酶合成基因 ,并对两种方法进行了比较。经IPTG诱导 ,相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 17%和 10 % ;不相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 39%和 2 0 %。共转化菌的酶活力比pET2 8caiB单质粒转化菌均提高 2 .3倍左右。两种双质粒转化系统相似 ,都需在外界抗生素选择压力下保持质粒稳定性     

S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成

旨在构建S-亚胺还原酶(S-IRED)和葡萄糖脱氢酶(GDH)在大肠杆菌中的一菌双酶共表达系统,实现辅酶NADPH的再生,高效合成手性仲胺。利用无缝克隆的手段设计构建一种单质粒双启动子共表达系统,以全细胞为催化剂催化手性仲胺S-2-甲基吡咯烷(S-2MP)的合成,并研究温度、pH及有机溶剂对双酶反应的影响。成功构建了S-IRED和GDH的重组共表达质粒,实现了S-IRED与GDH在大肠杆菌中的胞内共表达,以亚胺2-甲基吡咯啉(2MPN)为模式底物,以工程菌全细胞催化手性仲胺S-2MP的合成,在低辅酶添加时催化手性胺的产率和光学纯度均高于95%。该双酶共表达体系的最适温度和pH分别为37℃和pH 8,10%以下的甲醇对双酶反应有正向促进作用。大肠杆菌胞内双酶共表达系统的构建实现了辅酶NADPH的原位再生,降低了亚胺还原酶催化合成手性胺的成本,为手性胺的规模制备奠定了基础。     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

共表达TrxA和DsbC对富含二硫键的异源蛋白在大肠杆菌中表达的影响

以复制子为p15A的质粒pACU184为基础 ,构建了 3种表达硫氧还蛋白 (TrxA)或 和二硫键异构酶 (DsbC)的表达质粒 .经IPTG诱导 ,克隆的DsbC和TrxA都以可溶的形式高表达 .分别将构建的 3种表达质粒与复制子为colE1并克隆有人源化鼠抗人纤维蛋白单链抗体 低分子量尿激酶融合基因 (C6 UK)的表达质粒共转化大肠杆菌XL1 blue ,在 30℃用IPTG诱导表达 .SDS PAGE显示 ,共表达TrxA或DsbC都能导致C6 UK融合蛋白的部分可溶性表达 ,而且同时共表达TrxA和DsbC 2种分子时 ,C6 UK完全以可溶形式表达 ,但表达量降低 .分别用溶圈法和ELISA检测了各种共表达时可溶表达产物的生物活性 .结果显示 ,只有共表达DsbC时才能检测到明显的C6 UK融合蛋白的双功能     

GL-7ACA酰化酶表达检测系统的建立

戊二酰－7－氨基头孢烷酸（GL－7ACA）酰化酶能够催化GL－7ACA分解生成7－ACA,后者是工业半合成生产头孢类抗菌素所需的重要前体。为了准确地检测GL－7ACA酰化酶及其突变体的表达,本研究通过构建一系列质粒载体,建立了两个简便有效地测定GL－7ACA酰化酶基因acy表达量的系统,从而可对酶的比活力进行定量。我们将两个报告基因,即儿茶酚双加氧酶基因（xylE)和β－半乳糖苷酶基因（lacZ)分别置于acy基因的下游,使之与acy基因共用一个启动子,进行串联表达,各自构成一个多顺反子系统。实验证明,基因融合后的儿茶酚双加氧酶或β－半乳糖苷酶的活力可以间接反映acy的表达量。     

不相容双质粒共表达霍乱毒素类似嵌合蛋白

霍乱毒素由5个B亚基(CTB)和l个A亚基(CTA)(包括CTA1和CTA2)组成。该蛋白结构可帮助有毒的CTA1分子进入细胞。本研究拟利用原核不相容双质粒共表达系统获得霍乱毒素类似嵌合蛋白,用于大分子蛋白质黏膜给药的载体研究。将CTB基因片段克隆至载体p ET-28a中,获得重组质粒p ET-28a-CTB;以增强型绿色荧光蛋白(EGFP)代替有毒的CTA1,在CTA2序列N端融合穿膜肽(TAT),将EGFP-CTA2-TAT基因克隆至载体p ET-22b(+)中,获得重组质粒p ET-22b-EGFP-CTA2-TAT。利用p ET-28a-CTB和p ET-22b-EGFP-CTA2-TAT二者不同抗性,将双质粒分步转化到大肠杆菌BL21中。表达条件为0.75 mmol/L IPTG、20℃、200 r/min诱导20 h,重组嵌合蛋白能以可溶形式表达。经过Ni-NTA、Sephadex G-75纯化,Western blotting对蛋白质特异性进行鉴定,确定获得(CTB)5/EGFP-CTA2-TAT嵌合蛋白。     

共表达SHMT和TPase载体的构建及双酶法合成L-色氨酸

利用重组大肠杆菌表达丝氨酸羟甲基转移酶(SHMT)和色氨酸酶(TPase),并利用双酶法合成L-色氨酸。采用PCR从大肠杆菌K12基因组中扩增上述两种酶的基因,利用pET-28a载体,构建单表达重组质粒pET-SHMT、pET-TPase和共表达重组质粒pET-ST。将上述3种重组质粒转入大肠杆菌BL21(DE3)进行表达。SDS-PAGE结果表明,单表达基因工程菌BL21(DE3)/pET-SHMT和BL21(DE3)/pET-TPase分别在47kDa(SHMT)和50kDa(TPase)处有蛋白表达带;共表达基因工程菌BL21(DE3)/pET-ST在上述两处均有蛋白表达带。与宿主菌相比,单表达SHMT基因工程菌产酶活性提高了6.4倍;单表达TPase基因工程菌产酶活性提高了8.4倍;共表达SHMT和TPase基因工程菌产酶活性分别提高了6.1和6.9倍。利用工程菌所产酶进行双菌双酶法和单菌双酶法合成L-色氨酸。两菌双酶合成L-色氨酸的累积量达到41.5g/L,甘氨酸转化率为83.3%,吲哚转化率为92.5%;单菌双酶合成L-色氨酸的累积量达到28.9g/L,甘氨酸转化率为82.7%,吲哚转化率为82.9%。     

基于IRES序列的多基因共表达载体构建

目的:构建一个IRES序列介导的多基因共表达载体,实现两个目的基因和筛选标记基因共用一个启动子高效表达,提高多基因稳定共表达细胞株的筛选效率。方法:以实验室前期构建的载体pLV-MCS-Puro为骨架,设计并全基因合成双基因克隆表达元件,连接到骨架载体,构建多基因共表达载体pLV-2MCS-Puro,以DsRed2和EGFP荧光蛋白基因验证该载体用于多基因稳定共表达细胞株筛选的效率。结果:成功构建了pLV-2MCS-Puro载体以及DsRed2和EGFP共表达重组质粒pLV-DsRed2-EGFP-Puro。瞬时转染实验证明该载体能介导多基因共表达。抗性筛选获得了MDCK和HeLa两种细胞的多基因稳定共表达细胞池。细胞池涂片荧光显微镜观察和计数表明抗性细胞池DsRed2和EGFP双阳率接近100%。基因组和转录水平PCR及蛋白质免疫印迹实验表明,DsRed2和EGFP稳定整合到抗性细胞基因组,并且两种蛋白质表达水平较为一致。结论:成功构建了多基因共表达载体pLV-2MCS-Puro,实现了两个目的基因和抗性基因串联共表达,并且具有高效的多基因稳定共表达细胞株筛选效率。该载体在研究蛋白质相互作用及工程细胞构建等方面具有一定的应用前景。     

表达大肠杆菌分子伴侣GroEL、GroES和GrpE载体的构建及其应用

大肠杆菌（Escherichia coli）共表达系统常要求质粒具有不同抗生素抗性以及不同的复制子。利用粘性末端PCR技术,以含有大肠杆菌分子伴侣基因GroEL、GroES和唧E的pR—GESP质粒为模板,设计两对引物,通过两次独立的PCR反应扩增3个基因的多顺反子,将形成粘性末端的PCR产物插入NcoI和Xho1酶切的pACY．CDuet-1质粒,构建的pA—GESP质粒具有p15A复制子及氯霉素抗性,和具有ColE1复制子及卡那霉素抗性表达载体pET28b相容。SDS—PAGE显示含有pA—GESP质粒的大肠杆菌细胞中3个分子伴侣蛋白的表达水平和含有pR—GESP质粒的大肠杆菌细胞没有明显差异,它们对玉米丝氨酸消旋酶的可溶性表达有部分促进作用,但对N端含有组氨酸标签的玉米铁氧还蛋白还原酶的表达没有作用,在三个含有不同抗生素基因的质粒中共表达分子伴侣、5-氨基乙酰丙酸合酶和尿卟啉原III甲基化酶,两个酶连续催化的荧光产物在细胞内积累量为562．13±3．17／OD600,而没有分子伴侣的积累量为457．66±4．98／OD600,表明分子伴侣改善部分蛋白在大肠杆菌的可溶性表达和催化功能。     

基于分子伴侣策略提高NADPH依赖型醇脱氢酶的异源可溶性表达 *

目的:生物催化的氧化还原反应广泛应用于手性化合物的制备,其中很多反应涉及辅酶NADPH的原位再生。以异丙醇为辅助底物,利用醇脱氢酶再生NADPH,具有比酶活高、副产物丙酮易于分离等优势,受到越来越多的关注。选择极具应用潜力的来源于Clostridium beijerinckii的醇脱氢酶CbADH作为研究对象,针对其在大肠杆菌中的可溶性表达差、酶活低的瓶颈问题开展研究。方法: 首先通过引入诱导型质粒pGro7表达分子伴侣GroES-GroEL,将pET-28a(+)质粒表达CbADH的可溶性提高了3.57倍,酶活达到出发菌株的4.83倍。其次,考察了另外三种不同的分子伴侣表达策略:pET-28a(+)单质粒共表达、基因组强化表达GroES-GroEL和组成型改造pGro7/GroES-GroEL和pET-28a(+)/CbADH双质粒共表达。结果: 组成型改造pGro7和pET-28a(+)双质粒共表达策略的效果最优,其CbADH的可溶性表达提高了8.07倍,酶活达到了21.79U/mg DCW,是出发菌株的9.43倍。结论: 为CbADH的工业应用奠定了良好的基础,也为外源蛋白的可溶性表达提供了参考。     

A new method for protein coexpression in Escherichia coli using two incompatible plasmids

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.     

Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates

Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations.     

Monocyte chemotactic protein-1 secretion and expression after Toxoplasma gondii infection in vitro depend on the stage of the parasite

We constructed a series of plasmids that allow the insertion of cloned DNA in the Escherichia coli chromosome by site-specific integration into the bacteriophage HK022 bacterial attachment site. These plasmids make use of a ColE1 origin of replication, the phage HK022 attachment site attP, antibiotic resistance genes for selection and unique restriction sites. Circularisation of non-replicative fragments containing the HK022 attachment site attP is performed in vitro and site-specific integration of attP containing molecules is ensured by transfer into cells transiently expressing the HK022 integrase gene carried by a thermosensitive replicon. Insertion is very efficient and the inserted fragments are stably maintained without selection pressure. Since integrative fragments carry rarely used antibiotic markers conferring resistance to antibiotics hygromycin or apramycin, they can be used in most E. coli strains in conjunction with many replicative or integrative vectors.     

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.     

通过大肠杆菌thyA染色体-质粒平衡致死系统构建无抗性基因表达载体

克隆了大肠杆菌和霍乱弧菌胸腺嘧啶合成酶基因thyA ,并以pcDNA3质粒为基础 ,分别用两种来源的thyA基因替代其氨苄抗性基因Amp,构建了不含抗性基因 ,且可在thyA营养缺陷型大肠杆菌中基于染色体 质粒平衡致死系统稳定传代的真核表达载体。该载体可有效表达红色荧光蛋白报告基因。为核酸疫苗的制备提供一个无抗性的表达载体系统     

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

大肠杆菌ppsA和tktA基因的串联表达

ppsA和tktA是芳香族氨基酸生物合成中心途径的两个关键酶基因,在大肠杆菌中,ppsA基因编码磷酸烯醇式丙酮酸合成酶A(PpsA),该酶催化丙酮酸合成磷酸烯醇式丙酮酸;tktA基因编码转酮酶A,该酶在磷酸戊糖途径中生成4-磷酸赤藓糖起主要作用。采用PCR方法从大肠杆菌K-12株中扩增到ppsA和tktA,并实现了两基因的高效表达,其中ppsA活性提高了10．8倍,tktA活性提高了3．9倍,当这两个基因串联在一个质粒上导入大肠杆菌进行表达时,PpsA的活性变化较大(2．1～9．1倍),TktA的活性相对稳定(3．9～4．5倍),且这两个基因单独表达和串联表达都能使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量提高,且串联表达比单独表达较高。