大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

少棘蜈蚣候选杀虫肽κ-SLPTX-Ssm2e的原核表达与纯化

κ-SLPTX-Ssm2e是一条通过生物信息学同源序列分析获得的与已知具有杀虫活性的毒素κ-SLPTX-Ssm2a高度同源的少棘蜈蚣毒素,由31个氨基酸残基组成,且含有3对二硫键。为了后续便于对其结构和功能进行研究,现采用原核表达与纯化的方法来获取较高产量的毒素多肽。文中首先利用大肠杆菌自动诱导表达系统,在大肠杆菌表达菌株BL21(DE3)中表达融合蛋白GST-SUMO-κ-SLPTX-Ssm2e,将其通过GST亲和层析纯化后,采用Ulp1激酶酶切,以释放毒素分子。随后,通过反相高效液相色谱(RP-HPLC)进一步纯化。最后,利用MALDI-TOF/TOF质谱仪鉴定目的多肽峰的相对分子质量为3 475.732,与κ-SLPTX-Ssm2e的理论相对分子质量3 475.07基本一致,说明κ-SLPTX-Ssm2e很有可能被成功表达。上述结果为进一步研究κ-SLPTX-Ssm2e的杀虫活性奠定了基础。     

苏云金芽胞杆菌4.0718菌株中杀虫晶体蛋白基因的定位及鉴定

[目的]对苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)4.0718菌株中的杀虫晶体蛋白基因(insecticidal crystal protein gene,简称cry基因)进行定位和鉴定,系统分析高毒力Bt4.0718菌株的杀虫基因背景.[方法]采用脉冲电泳(PFGE)分离Bt 4.0718菌株的基因组DNA,确定该菌株的PFGE图谱和质粒图谱;采用Southern杂交分析该菌株中cry基因的定位,并使用PCR及PCR产物限制性片段长度多态性分析(PCR-RFLP)方法鉴定该菌株染色体和质粒上含有的cry基因类型.[结果]确定了Bt 4.0718菌株的PFGE图谱和质粒图潜,鉴定到Bt4.0718菌株的染色体和质粒上均定位有cry基因,且分别包含crylAa、crylAc、cry2Aa和cry2Ab 4种基因成分.但染色体上含有的cry基因可能不如质粒上含有的cry基因具有完整的开放阅渎框.[结论]首次在Bt 4.0718菌株的染色体上发现有丰富的cry基因,且与质粒上含有的cry基因类型一致.     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

重组人BMP6在大肠杆菌中可溶表达、纯化及活性分析

BMP6是一种调节成骨细胞和成软骨细胞分化的骨诱导因子,在修复各种骨缺损方面具有很好的应用潜力.有诱骨活性的BMP6是多二硫键的二聚体蛋白,疏水性极强容易聚集沉淀.为了在大肠杆菌中可溶表达具有生物活性的重组人BMP6(rhBMP6),构建了具有TRX、GST、MBP、CBD融合标签和His6标签的rhBMP6成熟肽原核表达载体,调节诱导温度和IPTG浓度,比较不同融合标签和诱导条件对目的蛋白表达量和溶解性的影响.结果表明,MBP最能有效的增强rhBMP6的溶解性,诱导条件对溶解性影响较小.大肠杆菌BL21 trxB(DE3)这种硫氧还蛋白还原酶缺陷菌株为rhBMP6二硫键在胞质中形成提供了合适的氧化还原环境.MBP和BL21 trxB(DE3)相结合在细胞质中高效可溶表达出了BMP6融合蛋白二聚体.表达产物经亲和层析和凝胶排阻层析纯化后,能诱导成肌细胞系C2C12向成骨细胞方向转化.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

Molecular cloning of an endoglucanase gene from an alkalophilic Bacillus sp. and its expression in Escherichia coli.

One of the cellulase genes from alkalophilic Bacillus sp. strain N-4 was cloned in pBR322. A recombinant plasmid, pYBC107, expressing carboxymethyl cellulase (CMCase) was isolated, and the size of the cloned HindIII fragment was found to be 5.5 kilobases. The restriction map of pYBC107 showed a different pattern from those of pNKI and pNKII (N. Sashihara, T. Kudo, and K. Horikoshi, J. Bacteriol. 158:503-506, 1984). When the HindIII fragment from pYBC107 was subcloned into pYEJ001, there was a 3.8-fold increase in CMCase activity over that observed with pYBC107. Plasmid pYBC108 constructed by treatment of pYBC107 with HindIII and EcoRI expressed the CMCase activity, although to a limited extent. To verify the originality of cloned pYBC107 from Bacillus sp., we analyzed the restriction digest by Southern blotting.     

Molecular cloning and expression of a xylanase gene from Cellulomonas sp. into Escherichia coli

Summary The xylanase gene of Cellulomonas sp. NCIM 2353 was cloned in pUC 18 and selected by growth on xylan as the sole carbon source. The functional clone harboured the recombinant plasmid with an insert of 1.42 kbp, as determined by restriction mapping and Southern hydridization. The clone secreted a xylanase of 45 000 mol. wt. as determined by Western blot analysis using specific antixylanase antibodies. The DNA insert carried the full structural gene along with its promoter and possibly regulatory sequences, since xylanase activity in the clone Cs11 was inducible by xylan. Offprint requests to: D. N. Deobagkar     

Molecular cloning and expression of cellulase genes of alkalophilic Bacillus sp. strain N-4 in Escherichia coli.

The genes for cellulases of alkalophilic Bacillus sp. strain N-4 were cloned in Escherichia coli with pBR322. Plasmids pNK1 and pNK2 were isolated from the transformants producing carboxymethyl cellulase, and the carboxymethyl cellulase genes cloned were in 2.0- and 2.8-kilobase-pair HindIII fragments, respectively. On the DNA level, the pNK1 fragment had a different restriction map from that of the pNK2 fragment, but the genomic hybridization experiments showed partial homology among these fragments. A total of 74 and 34% of the enzyme activities were observed in the periplasmic space of E. coli carrying the plasmids pNK1 and pNK2 , respectively. The carboxymethyl cellulase thus produced had broad pH activity curves (pH of 5 to 10.9) and was stable up to 75 degrees C.     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli.

The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.     

Molecular cloning of promoter-containing fragments from Bacillus stearothermophilus and their expression in Escherichia coli and Bacillus subtilis

Abstract Bacillus stearothermophilus DNA fragments containing a promoter were isolated in Escherichia coli using a shuttle promoter-probe vector. The molecular sizes of the isolated fragments ranged from 0.78 to 10 kb. The 0.78 and 1.1 kb fragments were selected and examined in some detail for promoter activity in both E. coli and Bacillus subtilis by analysis of expression of erythromycin-resistance (Em^r) and β-galactosidase. The results showed that the two fragments exhibit a high promoter activity in both bacteria. In vitro promoter activity of the 1.1 kb fragment was also shown by RNA syntheses catalyzed by RNA polymerases prepared from E. coli, B. subtilis and B. stearothermophilus .     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.     

Molecular cloning and expression in Escherichia coli of the Bacillus licheniformis bacitracin synthetase 2 gene.

The structural genes for the entire bacitracin synthetase 2 (component II) and for a part of the putative bacitracin synthetase 3 (component III) from Bacillus licheniformis ATCC 10716 were cloned and expressed in Escherichia coli. A cosmid library of B. licheniformis DNA was constructed. The library was screened for the ability to produce bacitracin synthetase by in situ immunoassay using anti-bacitracin synthetase antiserum. A positive clone designated B-15, which has a recombinant plasmid carrying about a 32-kilobase insert of B. licheniformis DNA, was further characterized. Analysis of crude cell extract from B-15 by polyacrylamide gel electrophoresis and Western blotting (immunoblotting) showed that the extract contains two immunoreactant proteins with high molecular weight. One band with a molecular weight of about 240,000 comigrates with bacitracin synthetase 2; the other band is a protein with a molecular weight of about 300,000. Partial purification of the gene products encoded by the recombinant plasmid by gel filtration and hydroxyapatite column chromatography revealed that one gene product catalyzes L-lysine- and L-ornithine-dependent ATP-PPi exchange reactions which are characteristic of bacitracin synthetase 2, and the other product catalyzes L-isoleucine-, L-leucine, L-valine-, and L-histidine-dependent ATP-PPi exchange activities, suggesting the activities of a part of bacitracin synthetase 3. Subcloning experiments indicated that the structural gene for bacitracin synthetase 2 is located near the middle of the insert.