红球菌Rhodococcus sp.DS-3 dszABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩(DBT)及其衍生物微生物脱硫的4S途径需要4个酶(DszA,DszB,DszC and DszD)参与催化。其中DBT单加氧酶(DszC or DBT-MO)和DBT-砜单加氧酶(DszA or DBTO2-MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2),FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA,DszB,DszC和DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA,DszB,DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%,3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。     

红球菌Rhodococcus sp. DS-3 dsz-ABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩（DBT）及其衍生物微生物脱硫的4S途径需要4个酶（DszA, DszB, DszC and DszD）参与催化。其中DBT单加氧酶(DszC or DBTMO)和DBT砜单加氧酶(DszA or DBTO2MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2), FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA, DszB, DszC 和 DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA, DszB, DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%, 3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。     

肝癌相关抗原SMP30重组基因的构建、表达及分子伴侣共表达系统的探索

旨在通过应用基因工程的方法构建、表达和纯化肝癌相关抗原SMP30,研究共表达分子伴侣提高基因工程蛋白表达的可溶性及效率。PCR扩增SMP30 cDNA序列,用基因工程技术构建重组表达质粒,转化E.coliBL21(DE3)pLysS宿主菌。表达蛋白经Ni-NTA亲和柱纯化获得HIS-SMP30融合蛋白;分别将4种表达不同分子伴侣的质粒(pG-KJE8、pGro7、pKJE7、pTf16)转入E.coliBL21(DE3)中;然后再将重组质粒转入含有分子伴侣质粒的细胞中,进行分子伴侣与重组质粒的共表达,SDS-PAGE检测目的蛋白的表达量与可溶性分析。经优化表达条件后,目的蛋白以包涵体形式表达,目的蛋白占总蛋白的60%以上;纯化后纯度高达95%以上;诱导共表达后,目的蛋白在上清含量极少,不到总表达目的蛋白的10%。成功构建出高效表达的SMP30重组质粒;加入到诱导表达体系中的4种分子伴侣质粒不能有效的促进可溶性蛋白的表达,pTf16共表达系统能增加目的蛋白表达量。     

弗氏柠檬酸菌甘油脱水酶激活因子基因的克隆、表达与功能鉴定

1,3-丙二醇是一种重要的化工原料,其生物法生产的研究逐渐受到的关注。研究以弗氏柠檬酸菌的总DNA为模板,通过PCR分别扩增出约1.8kb（dhaF）和0.4kb（dhaG）的两个基因片段分别编码甘油脱水酶激活因子大、小亚基, 连接于pMD-18T载体,测序分析显示与GenBank中相关基因的相似性最高为86%。将两基因以多顺反子的方式与pSE380连接构建表达载体,并在大肠杆菌中进行高效表达,表达量占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的甘油脱水酶激活因子,SDS-PAGE分析显示：大、小亚基分子量约为63kDa和12kDa;非变性胶分析显示：全酶的分子量约为150kDa,经扫描分析推测甘油脱水酶激活因子很有可能是以α2β2方式结合的。以弗氏柠檬酸菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能,为进一步阐明甘油脱水酶的激活机制及1,3-丙二醇的高效生产奠定了基础。     

鼠疫耶尔森氏菌质粒上重要毒力相关基因的克隆与表达

鼠疫耶尔森氏菌含有3种质粒pMT1、pPCP1和pCD1,这3种质粒编码鼠疫耶尔森氏菌的多种重要毒力因子。首先通过生物信息学技术选定了18种可能重要的毒力相关基因作为拟克隆和表达的目的基因。通过：PCR技术、TA克隆技术、双酶切技术获得目的片段。这些目的片段再分别克隆入原核表达载体pET32a中,构建了一系列重组表达质粒,其中12个重要的毒力相关基因在原核表达载体pET32a中有稳定的高效表达,表达量占细菌总蛋白的20％～40％。实验结果为进一步研究质粒编码的毒力因子的结构与功能,及其作为新型疫苗选择的可能性奠定了基础。     

纳豆激酶基因在E.coli HB101中的初步表达研究*

利用PCR技术以纳豆杆菌染色体DNA为模板扩增纳豆激酶基因 ,将该基因克隆到温度诱导型表达载体pBV2 2 0上 ,转化_{E .coli}HB1 0 1 ,获得转纳豆激酶基因重组菌。在确定了其最佳培养时间与诱导时间后 ,SDS PAGE分析结果表明基因表达产物为分泌型 ,蛋白表达量占菌体蛋白的12%左右 ,液体发酵后纳豆激酶产量可达 120U/mL菌液。对重组菌中重组质粒的稳定性进行研究 ,结果表明该质粒在宿主菌中具有良好的分离稳定性 ,而结构稳定性较差。     

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%。     

白鹅催乳素基因的克隆及诱导表达条件的优化

摘要: 运用RT-PCR方法, 从白鹅脑垂体总RNA中扩增得到了催乳素(Prolactin, PRL)基因编码区序列cDNA, 并将其克隆到pMD18-T载体上。DNA序列分析表明, PRL cDNA包括终止密码子在内的长度为690 bp,编码230个氨基酸残基的蛋白质, 与皖西白鹅的有所差异, 二者碱基同源性在99.57%, 氨基酸同源性达99.56%。将PRL基因编码区序列cDNA定向克隆到表达载体pET-32a (+)中, 构建表达质粒pET-32a(+)-PRL。该质粒的BL21 (DE3)转化菌在IPTG的诱导下可表达PRL基因融合蛋白, IPTG终浓度1 mmol/L, 37℃, 诱导4 h表达量最高, 表达量约占菌体总蛋白的28.96%。     

纳豆激酶基因在E.coli HB101中的初步表达研究

利用PCR技术以纳豆杆菌染色体DNA为模板扩增纳豆激酶基因,将该基因克隆到温度诱导型表达形体pVB220上,转化E．coliHB101,获得转豆激酶基因重组菌。在确定了其最佳培养时间与诱导时间后,SDS－PAGE分析结果表明基因表达产物为分泌型,蛋白表达量占菌体蛋白的12％左右,液体发酵后纳豆激酶产量可达120U／ml菌液,对重组菌中重组质粒的稳定性进行研究,结果表明该质粒在宿主菌中具有良好的分离稳定性,而结构稳定性较差。     

生长抑制激素基因在大肠杆菌中的表达

从pSom5中提纯生长抑制激素基因片段,在体外与大肠杆菌pBD_2质粒DNA重组连接,转化受体细胞D_2A_1,获得7株工程菌,在IPTG诱导下进行表达。采用β-半乳糖苷酶底物亲和层析法纯化,得到一纯净的分子量为50000道尔顿左右的蛋白质。对此蛋白质及其经CNBr化学裂解的产物进行放射免疫分析,证实化学合成的生长抑制激素基因已在大肠杆菌D_2,A_1中成功地表达,每升培养基中获得生长抑制激素41.69mg,占菌体总蛋白质的0.71%。     

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.     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

甘油脱水酶再激活酶的克隆表达及活性鉴定

运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

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     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration (c) 1993 Wiley & Sons, Inc.