牛种布鲁菌VirB12蛋白的原核表达及纯化

目的原核表达并纯化牛种布鲁菌(Brucella)VirB12蛋白。方法利用PCR法从牛种布鲁菌基因组中扩增VirB12基因,插入pET-30a(+)载体,构建重组表达质粒pETV12,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经组氨酸结合树脂柱纯化后,进行SDS-PAGE及Western blot分析。结果重组表达质粒pETV12经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约22 000,纯化的重组蛋白纯度达94%,可被布鲁菌免疫兔血清特异性识别。结论原核表达并纯化了牛种布鲁菌VirB12蛋白,为进一步研究VirB12蛋白的结构、功能及相关疫苗的研制奠定了基础。     

Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B12 Catalysis

Adenosylcobalamin (AdoCbl), or coenzyme B₁₂, is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co−C) bond for catalysis. How do the enzymes activate and cleave the Co−C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co−C bond and conformational stabilization of the adenosyl radical for coenzyme B₁₂ catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B₁₂ enzymes.     

大肠杆菌不耐热肠毒素B亚单位的原核表达及活性鉴定

目的克隆大肠杆菌不耐热肠毒素B亚单位(ltB)基因,构建其原核表达质粒,并对表达产物进行活性鉴定。方法采用PCR技术从产毒大肠杆菌129株基因组DNA中扩增ltB基因,克隆入载体pET-32a(+)中,构建原核表达质粒pET-32a(+)-ltB,转化E.coli BL21(DE3),IPTG诱导表达,Ni2+-NTA树脂层析柱进行纯化,纯化产物采用ELISA法鉴定其与牛GM1的结合活性。结果重组表达质粒经菌落PCR鉴定证明构建正确,所克隆的ItB基因与GenBank中报道的相应核苷酸序列的同源性为99.9%。重组菌诱导4h,目的蛋白表达量最高,约占菌体总蛋白的25%。纯化的重组LTB蛋白纯度约为96%,具有与牛GM1特异性结合的生物学活性。结论已成功克隆了ltB基因,并构建了其原核表达质粒,表达的重组蛋白具有一定的生物学活性。     

Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U‐turn and a looped‐out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U‐turn fold retained biological activity. An improvement of stacking interactions in the U‐turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U‐turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.     

维生素B12的技术进展

维生素B12是人和动物体内霞要的水溶性维生素之一。综述了维生素B12的性质、合成及生产情况。工业生产中,脱氮假单胞杆菌和费氏丙酸杆菌是主要的生产菌种,有厌氧和好氧2种生产工艺。今后应培养高产菌株,改进发酵工艺。     

牛布鲁菌L7/L12蛋白的原核表达及纯化

目的原核表达并纯化牛布鲁菌L7/L12蛋白。方法提取牛布鲁菌S19基因组DNA,PCR法扩增L7/L12基因,经测序正确后,克隆至pET-28a(+)载体,构建重组原核表达质粒pET-L7/L12,转化大肠杆菌BL21(DE3),IPTG诱导表达。SDS-PAGE分析目的蛋白的表达形式及表达量,经亲和层析纯化后,Western blot鉴定纯化产物。结果所构建的重组原核表达质粒pET-L7/L12经酶切鉴定表明构建正确。重组蛋白以包涵体形式表达,表达量占全菌总蛋白的33%;纯化的重组蛋白纯度可达94%,可与布鲁菌小鼠免疫血清发生特异反应。结论已成功表达并纯化了牛布鲁菌L7/L12蛋白,为建立高特异性的新型布鲁菌检测方法奠定了基础。     

Microbial and Genetic Resources for Cobalamin (Vitamin B12) Biosynthesis: From Ecosystems to Industrial Biotechnology

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.     

乙型肝炎病毒P蛋白在大肠杆菌中的分段表达及纯化

目的构建乙型肝炎病毒(HBV)P基因分段重组原核表达质粒,并进行目的蛋白的表达和纯化。方法PCR扩增HBVP基因的不同片段(1～534、1008～2040和2043～2526bp),分别经双酶切后克隆至原核表达载体pET-28a中,构建重组原核表达质粒,转化E.coliBL21(DE3),IPTG诱导表达,并对表达产物进行可溶性分析。用Ni2+-NTA凝胶亲和层析柱纯化融合蛋白,并进行Westernblot分析。结果重组原核表达质粒经酶切及测序证明构建正确。表达的融合蛋白主要以包涵体形式存在,表达量分别约占菌体总蛋白的34%、40%和36%。经Ni2+-NTA纯化的目的蛋白纯度达90%以上,且具有良好的反应原性。结论已成功构建了HBVP基因分段重组原核表达质粒,并在大肠杆菌中高效表达了重组蛋白,为研究HBVP蛋白的功能及病毒复制机制奠定了基础。     

汉坦病毒SEO型S基因的克隆及在大肠杆菌中的高效表达

目的构建汉坦病毒SEO型S基因原核表达载体。方法应用RT-PCR方法扩增汉坦病毒SEO型的YZG-Changchun株S基因,克隆至pMD18-T载体中,经酶切鉴定及PCR分析后,定向克隆入原核表达载体pET-28a中,转化E.coliRosetta,经IPTG诱导表达,SDS-PAGE和Western blot分析外源蛋白的表达情况。结果表达载体经双酶切和测序证明构建正确。IPTG浓度为1.0 mmol/L,诱导4.5 h时,S基因在原核细胞中得到了高效表达,表达的蛋白占菌体蛋白总量的37%,纯化后的蛋白具有良好的抗原活性。结论已成功构建了汉坦病毒SEO型S基因原核表达载体,并得到高效表达。     

重组人B淋巴细胞刺激因子工程菌生物学特性的稳定性

目的观察重组人B淋巴细胞刺激因子工程菌pKKH-BLyS/DH5α生物学特性的稳定性。方法工程菌连续传50代,每10代进行目的蛋白表达水平、质粒性状检测、透射电镜观察、革兰染色及各项生化检查,观察工程菌生物学特性的稳定性。结果0、10、20、30、40和50代工程菌之间目的蛋白的表达量无明显差异,基本维持在40%左右;双酶切、PCR及测序结果均与原代质粒相符;革兰染色均呈阴性;透射电镜观察及各项生化反应均呈现典型的大肠杆菌特性。结论该菌种生物学特性稳定,可作为生产用菌种。     

The Nitrogen Atom of Vitamin B6 Is Essential for the Catalysis of Radical Aminomutases

Radical aminomutases are pyridoxal 5′-phosphate (PLP, a B₆ vitamer)-dependent enzymes that require the generation of a 5′-deoxyadenosyl radical to initiate the catalytic cycle, to perform a 1,2 amino group shift reaction. The role of the nitrogen atom of PLP in radical aminomutases has not been investigated extensively yet. We report an alternative synthetic procedure to provide easy access to 1-deazaPLP (dAPLP), an isosteric analog of PLP which acts as a probe for studying the role of the nitrogen atom. Our results revealed that lysine 5,6-aminomutase (5,6-LAM), a radical aminomutase, reconstituted with dAPLP cannot turn over a substrate, demonstrating that the nitrogen atom is essential for radical aminomutases. In contrast, biochemical and spectroscopic studies on the S238A variant reconstituted with PLP revealed a minuscule loss of activity. This apparent anomaly can be explained by a water-mediated rescue of activity in S238A, as if mimicking the active site of lysine 2,3-aminomutase. This study leads to a better comprehension of how enzymes harness the optimum capability of PLP to realize catalysis.     

Characterization of polypyrrole/piperazine-1,4-bis(2-ethane sulfonate) film and its application for the immobilization of vitamin B12

Vitamin B12 (B12) - possessing a redox active cobalt centre - is a candidate to be used as a mediator in bioelectrochemical processes. In order to exploit the possible redox activity of B12, Pt has been modified by a bio-conform conducting layer polypyrrole/piperazine-1,4-bis(2-ethane sulfonate) PPy/PIPES. The electrochemical and spectral behaviour of this film proved to be similar to PPy/dodecyl-sulfate (DS), widely considered and accepted as one of the best combinations of conducting polymer films. The capability of the PPy/PIPES film for acting as adsorbant in the accumulation of B12 has been evidenced by the electrochemical quartz crystal microbalance (EQCM) technique. B12 could also be incorporated into the polymer layer during its electrochemical deposition. The results proving the preserved redox activity of B12 within the film open new perspectives towards redox mediated bioelectrochemical applications based on the immobilization of this biomolecule.     

重组人BAFF_(134～285)的克隆及在大肠杆菌中的高效表达

目的克隆人TNF家族的B细胞激活因子(BcellactivatingfactortotheTNFfamily,BAFF)胞外区cDNA,并进行高效表达和纯化。方法提取人新鲜扁桃体组织总RNA,经RTPCR扩增编码人BAFF胞外区134～285氨基酸残基cDNA,经序列测定后,克隆至pQE80L载体中,并转化大肠杆菌DH5α,经IPTG诱导表达及Ni2+NTA柱层析纯化目的蛋白,最后经SDSPAGE和Westernblot检测。结果RTPCR扩增得到了459bp的cDNA片段,序列分析与GenBank中报道的编码人BAFF134～285的cDNA序列一致,SDSPAGE及Westernblot证实表达蛋白确实为6×HisBAFF134～285融合蛋白,并存在于包涵体中。结论利用大肠杆菌可高效表达rhBAFF134285,为进一步研究其生物学活性奠定了基础。     

重组大肠杆菌不耐热肠毒素B亚单位表达条件的优化及其佐剂作用

目的优化重组大肠杆菌不耐热肠毒素B亚单位(LTB)的表达条件,并检测其黏膜佐剂作用。方法通过向LB培养基中添加葡萄糖(终浓度为0.5%)、低温培养诱导等方法诱导LTB的表达;采用阳离子交换法对目的蛋白进行纯化;以LTB作为佐剂进行免疫试验。结果可溶性重组大肠杆菌不耐热肠毒素(LTB)的表达量明显提高,纯化后蛋白含量达95%以上,经纯化的LTB作为三价流感裂解疫苗或gD抗原的佐剂免疫BALB/c小鼠,可提高小鼠血清及黏膜的抗体水平,且其效果与铝化剂及福氏佐剂相当或更高。结论优化了LTB的表达条件,且表达的LTB具有较强的黏膜免疫佐剂作用。     

Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.