A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P₁′ position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P₁′ position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.     

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.     

A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli

Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for ‘bacmid’ recombinants at 44°C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding β-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of 10⁵ per pg input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

代谢工程改造大肠杆菌合成己二酸

己二酸是一种具有重要应用价值的二元羧酸,是合成尼龙-66的关键前体。目前,生物法生产己二酸存在生产周期长、生产效率低的问题。本研究选择一株野生型高产琥珀酸菌株大肠杆菌(Escherichia coli) FMME N-2为底盘细胞,首先通过引入逆己二酸降解途径的关键酶,成功构建了可合成0.34 g/L己二酸的E. coli JL00菌株;接着,对合成路径限速酶进行表达优化,使E. coli JL01菌株在摇瓶发酵条件下产量达到0.87 g/L;随后,通过敲除sucD基因、过表达acs基因和突变lpd基因的组合策略平衡己二酸合成前体的供应,优化菌株E. coli JL12己二酸产量进一步提升至1.51 g/L;最后,在5 L发酵罐上对己二酸发酵工艺进行优化。工程菌株经72 h分批补料发酵,己二酸的产量达到22.3 g/L,转化率为0.25 g/g,生产强度为0.31 g/(L·h),具备了一定的应用潜力。本研究可为包括己二酸在内的多种二元羧酸细胞工厂的构建提供理论依据和技术基础。     

Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein.     

The stoichiometry of the ribosomal proteins of Escherichia coli

Summary A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.     

大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A（Disulfide bond formation protein A,DsbA）是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys³⁰残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys³⁰残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

Influence of process temperature on recombinant enzyme activity in Escherichia coli fed-batch cultures

The influence of proteolysis over recombinant protein quality has been studied using rhamnulose 1-phosphate aldolase (RhuA) production as case example. Progressive induction by means of continuous isopropyl-β-d-thiogalactopyranoside (IPTG) dosage in Escherichia coli fed-batch cultures led to high specific levels of recombinant protein. However, the specific activity profile did not correlate to the specific protein content when the process was run at 37 °C and there was a decrease of the enzyme activity along the induction phase. Specific activity loss depending on the presence of an energy source was observed at short term, but protein degradation due to the action of energy-independent metalloproteases occurred after a longer time period. The effects of lowering the temperature were analysed on both mechanisms, and a reduction of the specific activity loss was observed when the process temperature was decreased to 28 °C. Lower plasmid copy number and specific production rates probably alleviated the metabolic load on host cell during recombinant protein overexpression, and a high increase of the enzyme activity was achieved in high cell density fed-batch cultures under these conditions.     

Genetic studies of the ribosomal proteins in Escherichia coli X

Summary Episomes of E. coli, which cover argG but not the str region, were transferred to Serratia marcescens. Ribosomal proteins from these hybrid strains were analyzed with phospho-cellulose or carboxymethyl-cellulose column chromatography. Two E. coli ribosomal proteins, L21 and S15, could be detected in the ribosome from the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

Deuteration of Escherichia coli Enzyme I alters its stability

Enzyme I^Ntr is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI^Ntr. Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4 °C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI^Ntr are evaluated.     

Relaxation of supercoiled DNA associated with induction of heat shock proteins in Escherichia coli

Bacillus subtilis 168 is known to possess two thymidylate synthase (TSase; EC 2.1.1.45) genes: thyA and thyB. thyB encodes a thermosensitive TSase (inactivated at 46° C) which, in wild-type cells, accounts for only 5–8% of the total cellular TSase activity. In order to investigate the thermal lability of TSaseB we have analyzed the thyB genes of B. subtilis 168 and of an unrelated strain B. subtilis ATCC6633, which is shown here to have a temperature-resistant TSaseB. This conclusion is supported by the frequency of appearance of spontaneous Thy^– mutants at 37° C and 46° C, and by the analysis of clones containing the thyB genes from the two strains. The nucleotide sequences of these two thyB genes were compared.     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

Isolation of plasmids carrying the arginine repressor gene argR of Escherichia coli K12

Summary Twenty-two sexual crosses between strains of Phycomyces blakesleeanus carrying mutations affecting phototropism (madA, madD, madE), synthesis of carotenoids (carA), auxotrophy (leu-51, nicA, pur-51), and resistance to 5-fluorouracil (fur) were studied; mating type was also included as a marker. Recombination frequencies were obtained among the ten genes involved. Linkage was found between mating type and madE; leu-51 and madA; furA401, furB402 and madD. All other gene combinations tested are unliked.     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

[目的]为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略.[方法]将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒.将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基...     

大肠杆菌周质和外膜蛋白的定位

大肠杆菌周质和外膜蛋白发挥功能必须首先到达其特定亚细胞分区.大肠杆菌通过一系列与蛋白质分泌有关的蛋白（Sec蛋白）将周质和外膜蛋白转运至内膜.在切除了信号肽后,与周质蛋白的定位不同的是,外膜蛋白的最终定位还需要其他因子的协助.外膜蛋白的定位近来认为是以周质作为中介的.     

The process of general recombination in Escherichia coli K-12

Summary A review of the data on the genetic determination of general recombination in Escherichia coli introduces three alternative pathways of recombination, RecBC, RecF, and RecBCF. One recBC-dependent pathway is functional in recF ^– cells. An initiating endonuclease is involved, acting on the chi-sites of DNA. The second is recF-dependent, acting in the double mutant recBC sbcB. The corresponding endonuclease uses the fre-sites as a substrate. A third pathway acting in wild-type cells is mixed. Both enzymatic systems participate in the overall process. We shall call it RecBCF.Using the thermosensitive recA44 mutant it became possible to study the kinetics of integration of donor DNA into the recipient chromosome via the RecF and RecBCF pathways of recombination. The RecF pathway is characterized by delayed recombination; not less than 14 h being needed to complete the process at 35° C. By the RecBCF pathway (wild-type recipient) the reaction is fast, as described by Lloyd and Johnson (1979). The two stage nature of the RecF pathway is important. First an intermediate product is formed during a short time interval. This product is resistant to the degrading exonuclease V. Afterward the intermediate product is slowly integrated into the recipient chromosome. Autoradiography of this intermediate product, extracted from exconjugants, shows that it consists of closed DNA circles. Their length is within the limits 2–15 min on the E. coli map. Their average value is in fair agreement with genetic estimations of the integrated DNA fragments.Taking into consideration the similarity between genetic determinations of the fre-effects and the heterogeneity of the progeny, we conclude that the intermediate structures formed contribute to this heterogeneity.     

三种大肠杆菌表达的口蹄疫病毒A型多表位蛋白对猪的免疫原性比较

【目的】研制猪口蹄疫病毒(foot-and-mouth disease virus,FMDV)A型多表位蛋白疫苗,为猪FMDV A型的防控提供安全有效的疫苗。【方法】根据前期试验结果及国内外FMDVA型流行病学信息,设计并合成了3种多表位免疫原基因A10、IA10和FA10。在大肠杆菌BL21(DE3)中诱导表达,表达蛋白纯化复性后,制苗免疫猪。分别于免疫前和免疫后14和28d采血分离血清,用液相阻断ELISA(LPB-ELISA)方法检测血清IgG抗体滴度。免疫28d后用FMDV强毒攻毒,以评估免疫保护效果。【结果】SDS-PAGE和Western blotting结果证实A10、IA10和FA10三种蛋白均获得表达,分子量分别为35、57和64 kDa,与预测蛋白大小一致,且能被FMDV感染阳性血清所识别。LPB-ELISA结果表明,A10+201免疫组IgG滴度低于灭活疫苗组,但高于其他免疫组。攻毒后A10+201免疫组和灭活疫苗免疫组全部猪(5/5)获得保护,IA10+201和FA10+201免疫组80%(4/5)猪保护,A10和FA10免疫组只有20%(1/5)猪保护,而PBS+201组所有猪均未保护。【结论】A10+201免疫保护效果较好,可作为候选疫苗进行进一步评价。