重组蛋白融合信号肽在大肠杆菌周质空间表达的研究进展

重组蛋白在大肠杆菌中表达时,往往面临着形成包涵体的问题,而重组蛋白若是分泌至周质空间则基本解决了这一问题,周质空间的周质蛋白不仅能帮助重组蛋白正确折叠还有利于二硫键的生成。信号肽是一段由15-30个氨基酸组成,被融合在重组蛋白N端的短肽,按照结构、功能的不同可以划分为N区、H区和C区,具有引导重组蛋白转运至细胞周质空间的作用。本文综述了信号肽的结构组成、作用机理和基本分泌途径,讨论了信号肽的高效转运和筛选方法,总结了在大肠杆菌中重组蛋白融合信号肽实现周质表达的新进展,并对未来高效信号肽选择方面的研究进行了探讨。     

不同调控序列作用下GUS基因在烟草中瞬时表达活性的研究

以pBI121为出发质粒, 利用烟草泛素启动子Ubi.U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片, 检测瞬时表达活性, 研究不同调控序列对外源基因表达的调控作用。结果表明: CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍; 双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当; 烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1.5倍; Ubi.U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高, 其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍, 为CaMV35S独立作用时的10倍。     

β干扰素基因在大肠杆菌中的表达

通过Bal 31酶酶切及筛选从β干扰素基因组克隆中分离到成熟β干扰素基因,并组建了一个在Tac启动子控制下表达β干扰索基因的质粒。研究了不同诱导条件对β干扰素表达的影响。Β干扰素在大肠杆菌中表达的产量为10μ／l。     

不同调控序列作用下GUS基因在烟草中瞬时表达活性

以pB1121为出发质粒,利用烟草泛素启动子Ubi．U4、CaMV35S启动子以及Kozak序列构建4种GUS基因表达载体,通过叶盘转化法转化烟草叶片,检测瞬时表达活性,研究不同调控序列对外源基因表达的调控作用。结果表明：CaMV35S启动子附加Kozak序列后使GUS活性比独立使用CaMV35S提高了近2倍：双CaMV35S启动子附加Kozak序列驱动GUS基因的表达活性与单CaMV35S附加Kozak序列相当;烟草泛素启动子附加Kozak序列的表达活性为CaMV35S启动子附加Kozak序列的1．5倍;Ubi．U4-CaMV35S复合启动子附加Kozak序列驱动GUS基因表达水平最高,其表达效率是双CaMV35S启动子附加Kozak序列调控下GUS表达效率的3倍,为CaMV35S独立作用时的10倍。     

不同启动子驱动下转基因盐藻外源基因的稳定表达

摘要：为了探讨外源性与内源性启动子对转基因盐藻外源基因表达的影响,将含外源性启动子CMV35S的表达载体CMV35S-bar（G12）和含内源双拷贝碳酸酐酶启动子DCA1的表达载体DCA1-bar（D-B）分别转化盐藻,筛选稳定转化株后,观察在不同启动子驱动下外源基因的表达情况及对转基因盐藻生长的影响。 通过电击法分别将表达载体G12 和D-B转化盐藻,经PPT筛选后,各得到了3株PPT抗性藻株,经PCR及测序分析证实外源基因bar已经整合到盐藻的基因组中,半定量RT-PCR结果显示,在内源性启动子DCA1驱动下,bar基因的表达强度明显高于在外源性启动子驱动下bar的表达,并且D-B转化株的bar基因表达在盐诱导下其表达明显提高,而G12转化株中bar基因的表达对盐诱导无反应。Southern blot 分析显示,外源基因的拷贝数与不同启动子间无相关性。转化株的生长特性分析显示,D-B转化株的生长速度明显高于G12转化株。本研究的结果指出,内源诱导型启动子在驱动转基因盐藻外源基因的高效稳定表达中比外源组成型启动子更具有优势。     

产纤维素酶大肠杆菌工程菌的构建和酶的表达

为了使大肠杆菌(Escherichia coli)具备生产纤维素酶的能力,从铜绿假单胞菌(Pseudomonas aeruginosa)PKC-001克隆碱性纤维素酶基因,进行密码子优化后连到载体pET-22b,转入大肠杆菌BL21(DE3)菌株,对工程菌进行表达优化并分析纤维素酶酶活。结果表明大肠杆菌工程菌能生产分子量约39 kD的纤维素酶,在培养温度20℃、摇床转速170 r/min、IPTG终浓度0.01 mmol/L的条件下,纤维素酶的产量最大;在酸性条件下,细胞内和细胞外的滤纸酶活力(FPA)分别为5.78 U/mL和1.13 U/mL,在碱性条件下检测不到滤纸酶活。显然,重组纤维素酶仅在酸性条件下有活力。     

里氏木霉内切-β-葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质研究

采用外显子拼接的方法,以里氏木霉Trichoderma reesei基因组 DNA 为模板,克隆出内切-1,4-β-D-葡聚糖酶II基因egl2的全编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pQY2025。重组质粒线性化后用电穿孔法导入毕赤酵母Pichia pastoris菌株GS115中,经大量筛选,获得高效分泌表达内切葡聚糖酶II的毕赤酵母工程菌株Gp2025。用甲醇诱导培养基进行摇瓶发酵,培养基中内切葡聚糖酶II的活力可达1573.0U/mL,同时对重组内切葡聚糖酶II的性质进行了初步研究。     

信号肽对短杆菌胆固醇氧化酶基因在大肠杆菌中表达的影响

从Brevibacterium sp．DGCDC-82染色体DNA中扩增出含信号肽序列的胆固醇氧化酶结构基因,插入大肠杆菌表达载体pET28a（＋）中,构建重组质粒pET28a—COD（s＋）。以pET28a-COD（s＋）为底物,扩增出不含信号肽的胆固醇氧化酶结构基因,构建成重组质粒pET28a-COD（s-）。两种重组载体转化大肠杆菌BL21（DE3）通过IPTG诱导均获得活性表达,SDS-PAGE分析,目的产物表达量都占到了细胞总蛋白的50％以上,Brevibacterium sp．DCR2DC-82胆固醇氧化酶的信号肽对重组酶的空间构象和表达量并没有太大的影响。     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响

枯草芽孢杆菌具有良好的安全性和优异的外分泌能力,是目前常用的脂肪酶异源分泌表达体系。在枯草体系中如何提高脂肪酶分泌量是目前的研究热点。该体系中信号肽与目的基因不匹配和目标蛋白总表达量不高是导致脂肪酶LipS分泌表达效果较差的重要原因。针对这些问题,依据信号肽结构特征的不同(正电荷与疏水氨基酸个数的差异)对枯草芽孢杆菌体系中Sec和Tat这两个主要分泌途径的信号肽进行了筛选,结果显示Tat途径中的phoD信号肽分泌效果明显优于其他信号肽。在此基础上,通过替换使用诱导型表达载体、突变优化phoD信号肽,并考察了表达条件的影响,显著提高了脂肪酶LipS的分泌表达量,发酵液中转酯酶活达到62.07U/L,占总酶活的62.30%,较原始分泌表达体系的分泌量提高了13.7倍。     

Export of Erwinia chrysanthemi (EC16) protease by Escherichia coli

Abstract During exponential growth, Erwinia chrysanthemi (EC16) exports 99% of the protease (PRT) into the growth medium. By screening an EC16 genomic library in Escherichia coli HB101, several Prt⁺ clones were identified. A 16-kb Eco RI fragment, carrying the prt gene, was subcloned into pBR322 (pAKC326). E. coli HB101[pAKC326] cells exported PRT into the growth medium during exponential growth. PRT export was not accompanied by periplasmic leakage. E. coli HB101 carrying EC16 prt and pel genes (encoding pectate lyase) exported PRT but retained PEL in the periplasm. These findings indicate the occurrence of a PRT-specific export system in EC16, which is also functional in an E. coli strain carrying the prt ⁺ DNA segment.     

Flavohaemoglobin HmpX from Erwinia chrysanthemi confers nitrosative stress tolerance and affects the plant hypersensitive reaction by intercepting nitric oxide produced by the host

Host cells respond to infection by generating nitric oxide (NO) as a cytotoxic weapon to facilitate killing of invading microbes. Bacterial flavohaemoglobins are well-known scavengers of NO and play a crucial role in protecting animal pathogens from nitrosative stress during infection. Erwinia chrysanthemi, which causes macerating diseases in a wide variety of plants, possesses a flavohaemoglobin (HmpX) whose function in plant pathogens has remained unclear. Here we show that HmpX consumes NO and prevents inhibition by NO of cell respiration, indicating a role in protection from nitrosative stress. Furthermore, infection of Saintpaulia ionantha plants with an HmpX-deficient mutant of E. chrysanthemi revealed that the lack of NO scavenging activity causes the accumulation of unusually high levels of NO in host tissue and triggers hypersensitive cell death. Introduction of the wild-type hmpX gene in an incompatible strain of Pseudomonas syringae had a dramatic effect on the hypersensitive cell death in soya bean cell suspensions, and markedly reduced the development of macroscopic symptoms in Arabidopsis thaliana plants. These observations indicate that HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death programme.     

Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi

Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source. All the strains used survived immobilization into k-carrageenan beads. Immobilized E. chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension. Carrageenan immobilization techniques could provide a system to mimic the conditions of E. chrysanthemi cells in the infected plant tissue. This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 215–219. Received 04 April 2001/ Accepted in revised form 12 June 2001     

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kd_app for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.     

Beta-aspartylpeptides as substrates of L-asparaginases from Escherichia coli and Erwinia chrysanthemi

L-Asparaginase is known to catalyze the hydrolysis of L-asparagine to L-aspartic and ammonia, but little is known about its action on peptides. When we incubated L-asparaginases purified either from Escherichia coli or Erwinia chrysanthemi - commonly used as chemotherapeutic agents because of their antitumour activity - with eight small beta-aspartylpeptides such as beta-aspartylserineamide, beta-aspartylalanineamide, beta-aspartylglycineamide and beta-aspartylglycine, we found that both L-asparaginases could catalyze the hydrolysis of five of them yielding L-aspartic acid and amino acids or peptides. Our data show that L-asparaginases can hydrolyze beta-aspartylpeptides and suggest that L-asparaginase therapy may affect the metabolism of beta-aspartylpeptides present in human body.     

Expression of Erwinia chrysanthemi ENA49 uvr gene in Escherichia coli K12 cells

The uvrA gene of Erwinia chrysanthemi ENA49 similar to uvrA gene of Escherichia coli K12 has been cloned in vivo in Escherichia coli AB1886 uvrA6 cells using the plasmid pULB113 (RP4mini Mu). The presence of pULB113 carrying uvrA gene of Erwinia in Escherichia coli K12 uvrA- cells resulted in suppression of this mutation while uvrB and uvrC are not suppressed by this locus. The genetic control of excision repair of UV-damage in Erwinia chrysanthemi ENA49 is concluded to be similar to the one in Escherichia coli K12.     

Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.