重组大肠杆菌可溶表达并快速定量GFP-hepA融合蛋白

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.     

大肠杆菌中表达来源于Agrobacterium radibacter的新hamA基因以生产5-氨基乙酰丙醇

A new hemA gene encoding 5-aminolevulinate （ALA） synthase was cloned from Agrobacterium radiobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coenzyme A （succinyl-CoA） and glycine to produce ALA. Four plasmids carrying the A. radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 （DE3） harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside （IPTG） addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8 nmol.min^-1.mg^-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9g.L^-1.     

补料分批培养耐乙酸大肠杆菌生产人表皮生长因子

An acetate-tolerant mutant of Escherichia coli DH5α, DA19, was used for secretory production of human epidermal growth factor （hEGF） whose expression was under the control of phoA promoter. The recombinant cells were cultured in a chemically defined medium, and glucose was added at different specific provision rates during the growth and expression phases. It was found that pH had a significant effect on the extracellular hEGF production. The extracellular hEGF concentration was 75.5mg·L^-1, 5.2-fold of the level reached at pH 7.0, even though more acetate was produced. Nitrogen source was limited in the later growth phase. Supplementation of ammonium promoted the consumption of phosphate and reduced the time to exhaust phosphate, but the extracellular hEGF production was similar to that without supplementation of ammonium.     

运用试验设计方法提高胞外杂合b-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L^-1. After optimization of the medium, 297.71U·ml^-1 of β-glucanase activity in the medium and 352350U·g^-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

运用试验读者设计方法提高胞外杂合β-葡聚糖酶在重组大肠杆菌中的表达

A genetically engineered Escherichia coli JM109 harboring pLF3 was used to produce a hybrid extracellular β-glucanase. Starting with enzyme production medium, glycerol and yeast extract combined with NaNO3 were screened to be the most suitable carbon and nitrogen source, respectively. Analysis of six components of the enzyme production medium by employing statistical optimization methods such as Plackett-Burman design and steepest ascent showed that yeast extract was the only significant variable and its best concentration for enzyme production was 12g·L-1. After optimization of the medium, 297.71U·ml-1 of β-glucanase activity in the medium and 352350U·g-1 of β-glucanase selectivity could be obtained, which were 14 and 72 folds higher than those obtained from original medium, respectively. Even higher enzyme activities were achieved by batch cultivations in a conventional stirred bioreactor on the optimized medium.     

重组大肠杆菌高密度发酵生产类人胶原蛋白的过程控制研究

Recombinant E. coli BL 21 was cultivated in high cell density to produce human-like collagen. The effects of the feeding of nitrogen source, controlled by an auto on/off-feeding mode with two different cycles of 0.5min and 4min intervals, oxygen-enrichment methods and inducement strength on the cell yield and human-like collagen production were investigated. The studies showed that nitrogen source feeding in fast cycle could result in higher human-like collagen production than that in slow cycle; and the feedback regulation of glucose, increase of the pressure of fermentation bioreactor, and supply of oxygen-enriched air could all increase cell yield and human-like collagen production. The effects of inducement strength on protein expression were found important. When OD600 reached 90-100, the cultivation temperature was increased to 42℃ to begin induction for 2-3 h, and then shifted to 39℃ for 5-6h induction, the cell density and human-like collagen production could reach 96g·L-1 [DCW (dry cell mass)     

Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q10

The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal in-tegration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E. coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution （multiple copy integration） and replicon-free and markerless chromosomal integration （single copy integration）, respectively. A coenzyme Q10 hyper-producer Es-cherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway. The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass （DCM） of coenzyme Q10 when supplemented with 0.075 g·L^-1 of 4-hydroxy benzoic acid;this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.     

基因敲除谷氨酸棒杆菌提高鸟氨酸产量及比较蛋白质组学分析(英文)

Engineered Corynebacterium glutamicum was constructed for L-ornithine production by disrupting genes of argF and proB to prevent the flux away from L-ornithine.Effect of the inactivation of 2-oxoglutarate de-hydrogenase complex(ODHC) on L-ornithine production was also investigated.It was found that the inactivation of ODHC by knockout of the kgd gene enhanced L-ornithine production.The engineered C.glutamicum ATCC13032(ΔargFΔproBΔkgd) produced L-ornithine up to 4.78 g·L-1 from 0.24 g·L-1 of the wild-type strain.In order to understand the mechanism of L-ornithine production in C.glutamicum ATCC13032(ΔargFΔproBΔkgd) and find out new strategies for further enhancing L-ornithine production,the comparative proteome between the wild-type and the engineered strain was analyzed.L-Ornithine overproduction in the engineered strain was related to the up-regulation of the expression levels of enzymes involved in L-ornithine biosynthesis pathway and down-regulation of the expression levels of proteins involved in pentose phosphate pathway.The overexpression of genes in the upstream pathway of glutamate to increase the availability of endogenous glutamate may further in-crease ornithine production in the engineered C.glutamicum and the ornithine synthesis enzymes(ArgCJBD) may not be the limiting enzymes in the engineered C.glutamicum.     

甲醇营养型重组毕赤酵母高效表达外源蛋白过程的控制与优化(英文)

The methylotrophic yeast Pichia pastoris is a highly successful system for production of a variety of heterologous proteins due to its unique features/abilities for effective protein expression, and tremendous efforts have been made to increase heterologous protein productivity by P. pastoris in recent years. When new engineered yeast strains are constructed and are ready to use for industrial protein production, process control and optimization techniques should be applied to improve the fermentation performance in the following aspects: (1) increase recombinant cell concentrations in fermentor to high density during growth phase; (2) effectively induce heterologous proteins by enhancing/stabilizing titers or concentrations of the proteins during induction phase; (3) decrease operation costs by relieving the working loads of heat-exchange and oxygen supply. This article reviews and discusses the key and commonly used techniques in heterologous protein production by P. pastoris, with the focus on optimizations of fermentation media and basic operation conditions, development of optimal glycerol feeding strategies for achieving high density cultivation of P. pastoris and effective heterologous protein induction methods by regulating specific growth rate, methanol concentration, temperatures, mixture ratio of multi-carbon substrates, etc. Metabolic analysis for recombinant protein production by P. pastoris is also introduced to interpret the mechanism of sub-optimal heterologous protein production and to explore further optimal expression methods.     

多策略代谢工程大肠杆菌高效生产辅酶Q10

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli (△ispB::ddsA,△pykFA and △gapA::gapC),harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.     

人角质细胞生长因子的克隆及表达

目的为获得高表达的重组人角质细胞生长因子生物工程菌。方法应用ＲＴ－ＰＣＲ技术,从人胚 胎肺成纤维细胞中,扩增出ＫＧＦ ｃＤＮＡ基因,并插入ｐＵＣ１８－Ｔ载体,构建成了重组人ＫＧＦ基因,经ＤＮＡ测序证实与 文献报道一致。将ＫＧＦ基因定向插入大肠杆菌表达载体ＰＥＴ１１ｂ内,转化大肠杆菌ＨＢ１０１。结果获得的重组子 经ＩＰＴＧ诱导,在相对分子质量为１９０００处表达重组蛋白,约占菌体蛋白的１０％。结论建立了制备重组人ＫＧＦ表 达系统,为今后进一步研究ＫＧＦ的生物学功能奠定可靠的物质基础。     

人表皮生长因子受体HER2胞外近膜区基因的克隆及原核表达

目的克隆人表皮生长因子受体HER2胞外近膜区基因,原核表达并纯化重组蛋白。方法从人乳腺癌细胞系SK-Br3中扩增HER2胞外近膜区编码基因,克隆并测序后,插入原核表达质粒pET41d中,转化大肠杆菌BL21(DE3),IPTG诱导表达,并进行纯化。结果从SK-Br3细胞系中扩增出387bp的人HER2胞外近膜区基因;重组表达质粒pET41d/HER2构建正确;IPTG浓度为0.5mmol/L时,目的蛋白的表达量最高;纯化后重组蛋白浓度为1.5mg/ml。结论已成功表达了HER2胞外近膜区蛋白,为抗HER2单克隆抗体的制备提供了抗原。     

乙型脑炎病毒SA14-14-2株NS3基因的克隆及原核表达

目的克隆并表达乙型脑炎病毒SA14-14-2株NS3编码区的基因片段,为重组蛋白进一步的功能研究奠定基础。方法提取乙脑病毒SA14-14-2株总RNA,用RT-PCR法扩增NS3-1、NS3-2基因片段,克隆入原核表达载体pET15bTAT中,构建重组原核表达质粒,转化大肠杆菌Rosetta2,IPTG诱导表达。表达产物经Ni2+亲和层析柱纯化后,进行Western blot鉴定。结果重组原核表达质粒pET15bTAT-NS3-1和pET15bTAT-NS3-2经酶切证明构建正确。表达的重组蛋白相对分子质量约为27000和20000,主要以包涵体的形式表达。重组蛋白纯化后纯度可达80%以上,并可被小鼠抗乙型脑炎病毒SA14-14-2株血清识别。结论已成功克隆了乙型脑炎病毒SA14-14-2株NS3-1和NS3-2基因,并在大肠杆菌Rosetta2中获得表达。     

小鼠可溶性IL-5受体α胞外区基因的克隆及原核表达

目的克隆并原核表达小鼠可溶性IL-5受体α(sIL-5Rα)胞外区基因。方法采用RT-PCR从BALB/c小鼠脾脏组织中分别扩增sIL-5Rα胞外区前后段基因片段,酶切后将两段基因连接,插入原核表达载体pPROEX中,构建重组表达质粒pPROEX/sIL-5Rα,转化大肠杆菌DH5α,IPTG诱导表达。采用SDS-PAGE和Western blot检测目的蛋白的表达。结果重组表达质粒经双酶切和DNA测序,证实构建正确。表达的sIL-5Rα胞外区蛋白相对分子质量约36100,表达量约占菌体总蛋白的30%,且可与兔抗小鼠sIL-5Rα单抗发生特异性免疫反应。结论已成功克隆并原核表达了小鼠sIL-5Rα胞外区基因,为进一步探讨sIL-5Rα对哮喘的治疗作用及开发新的哮喘治疗药物奠定了基础。     

HIV-1 HXB2株Tat核心碱性区多肽Tat_(38-61)的原核表达及其免疫反应性

目的 构建HIV-1 Tat核心碱性区多肽Tat38-61重组原核表达质粒,在大肠杆菌中表达融合蛋白并进行纯化及免疫反应性检测。方法采用PCR法从HIV-1 HXB2株Tat1-101基因中扩增编码Tat38-61的基因序列,克隆至原核表达载体pET32a(+)中,构建重组原核表达质粒pET32a(+)-Tat38-61。转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经Ni2+-NTA柱亲和层析法纯化后,ELISA法鉴定其免疫反应性。结果重组原核表达质粒pET32a(+)-Tat38-61经双酶切及测序表明构建正确;SDS-PAGE分析显示,在相对分子质量约21 300处可见目的 蛋白条带,表达量占菌体总蛋白的67.4%,主要以可溶形式表达;纯化后融合蛋白的纯度可达97%以上;ELISA结果显示,该融合蛋白与兔抗PEPTIDE-Tat1-101血清及HIV阳性血清均呈特异性反应。结论已成功构建了HIV-1 Tat核心碱性区多肽Tat38-61的重组原核表达质粒,表达并纯化了PET32a(+)-Tat38-61融合蛋白,该融合蛋白碱性区表位得到较好的保留,为Tat38-61噬菌体突变文库的构建及亲和筛选奠定了基础。     

新型异亮氨酸双加氧酶及其重组大肠杆菌合成羟基异亮氨酸

异亮氨酸双加氧酶（IDO）可特异性的转化底物L-异亮氨酸（L-Ile）生成4-羟基-L-异亮氨酸（4-HIL）,该产物具有促进胰岛素分泌的功能,可用于抗糖尿病、降胆固醇等。本研究结合了酶标显色和薄层层析（TLC）的方法从自然界中筛到了具有IDO活性的菌株,并将该菌株中的目的基因ido克隆到大肠杆菌中,获得重组表达菌株,并且验证该菌具有IDO的转化功能。本研究优化了转化反应体系和条件,同时通过30℃过夜温育菌体细胞的方法,使该菌株全细胞转化合成4-HIL的产率达到85%以上。     

Reconstruction of tyrosol synthetic pathways in Escherichia coli

Tyrosol is a pharmacologically active phenolic compound widely used in the medicine and chemical industries. Traditional methods of plant extraction are complicated and chemical synthesis of tyrosol is not commercially viable. In this study, a recombinant Escherichia coli strain was constructed by overexpressing the phenylpyruvate decarboxylase ARO10 from Saccharomyces cerevisiae, which could produce tyrosol from glucose. Furthermore, genes encoding key enzymes from the competing phenylalanine and tyrosine synthesis pathways and the repression protein TyrR were eliminated, and the resulting engineered strain generated 3.57 mmol·L^-1 tyrosol from glucose. More significantly, codon optimization of ARO10 increased expression and tyrosol titer. Using the novel engineered strain expressing codon-optimized AR10 in shake-flask culture, 8.72 mmol·L^-1 tyrosol was obtained after 48 h. Optimization of the induction conditions improved tyrosol production to 9.53 mmol·L^-1 (1316.3 mg·L^-1). A higher titer of tyrosol was achieved by reconstruction of tyrosol synthetic pathway in E. coli.     

百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达

目的克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白。方法从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆入载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经8mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性。结果重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合。结论已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础。     

Effects of nutrients on α-amyiase production and plasmid stability in fed-batch culture of recombinant bacteria

Escherichia coli harbouring a recombinant plasmid containing the α-amylase gene (HB101/pHI301) and its derivative which can stably maintain the plasmid (HB101/pHI301A) were cultivated in a mini-jar fermenter. By controlling dissolved oxygen (DO) at a concentration of 2–3 μg/l and supplementing with suitable amounts of nutrients, the organisms could grow to a concentration (up to 40 g dry cell l^?1) in a semi-synthetic casamino acid medium. During culture the plasmid was maintained stably in both strains. In a synthetic medium, both micro-organisms grew less well and plasmid-free segregants were observed with HB101/pHI301 during the fed-batch culture. On the other hand, plasmid pHI301A was stably maintained throughout the fed-batch culture of HB101/pHI301A. Growth inhibitors, which may be metabolic products, accumulated in the medium and reduced bacterial growth.