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1.
Synthesis of rat transferrin in Escherichia coli containing a recombinant bacteriophage 总被引:1,自引:0,他引:1
A R Aldred G J Howlett G Schreiber 《Biochemical and biophysical research communications》1984,122(3):960-965
Using mRNA from rat liver a cDNA library was constructed in lambda gt11Amp3. Immunochemical screening identified 15 clones producing transferrin. The identity of two clones was confirmed by nucleotide sequencing, which also indicated a presegment rich in hydrophobic amino acids but lack of a prosegment in precursor transferrin. A 920 base pair insert in one clone corresponded to 84% of the N-terminal domain of transferrin, which was synthesized as a hybrid protein with bacterial beta-galactosidase. A 1540 base pair insert in another clone corresponded to the N-terminal plus 50% of the carboxy terminal domain of transferrin. The product of this clone possessed only antigenic properties of transferrin. 相似文献
2.
The synthesis of chiral cyanohydrins by oxynitrilases 总被引:6,自引:0,他引:6
Enantiomerically pure cyanohydrins are important synthetic intermediates for pharmaceuticals and agrochemicals. They are produced by enzyme-catalysed synthesis using oxynitrilases. Sufficient quantities of enzyme are available via cheap natural sources and there have been recent advances in overexpression production of cyanohydrins on an industrial scale. 相似文献
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Summary The recombinant Escherichia coli strain, TG2 (p1/1), can be used for biotransformation of toluene to toluene cis-glycol. However, toluene caused substantial inhibition of both growth and toluene cis-glycol production at concentrations above 0.04%. Tetradecane was found to be non-toxic at a phase ratio of 0.23 and provided protection against toluene toxicity. Thus, growth was still possible in the presence of 1.5% toluene. Furthermore, the yield of toluene cis-glycol was increased 1.7-fold compared with the yield in the absence of tetradecane. 相似文献
6.
F A de Jong G Howlett A R Aldred N Fidge G Schreiber 《Biochemical and biophysical research communications》1984,119(2):657-662
A cDNA library was constructed from rat liver polyadenylated RNA using the expression vector lambda gt11-Amp3. Several clones expressing antigenic determinants for rat apolipoprotein E were identified. The cDNA insert in one clone was further characterized and found to have a sufficient length (1120 base pairs) to code for full length apolipoprotein E. Restriction mapping and nucleotide sequencing showed the clone to contain the coding region for apolipoprotein E flanked by about 120 nucleotides at the 3'-side and by about 64 nucleotides on the 5'-side. One of the proteins produced by the clone was found to be a prokaryotic/eukaryotic hybrid protein reacting with antibodies to both bacterial beta-galactosidase and rat apolipoprotein E. 相似文献
7.
《Journal of Molecular Catalysis .B, Enzymatic》2011,71(3-4):114-118
(S)-3-Chloro-1-phenyl-1-propanol ((S)-CPPO) is a useful chiral building block for the synthesis of anti-depressant drugs. The yeast reductase, YOL151W, evidences enantioselective reduction activity, converting 3-chloro-1-phenyl-1-propanone (3-CPP) into (S)-CPPO. Escherichia coli whole cells co-expressing YOL151W and Bacillus subtilis glucose dehydrogenase were employed for the synthesis of CPPO following permeabilization treatment. A reaction system employing these recombinant E. coli whole cells could convert 30 mM 3-CPP enantioselectively into (S)-CPPO. In an effort to enhance substrate solubility and to prevent substrate/product inhibition during the enzyme reaction process, a variety of ionic liquids were tested and [Bmim][NTf2] was ultimately selected for the ionic liquid/water two phase system. Tween 40 was added to accomplish the efficient mixing of the two phases. Using these recombinant E. coli whole cells and the [Bmim][NTf2]/water two phase system, 100 mM (S)-CPPO was generated with an enantiomeric excess of >99%. 相似文献
8.
Taran SA Verevkina KN Esikova TZ Feofanov SA Miroshnikov AI 《Prikladnaia biokhimiia i mikrobiologiia》2008,44(2):181-186
Cladribine (2-chloro-2'-deoxyadenosine) was synthesized using intact cells of the recombinant Escherichia coli strain producing Geobacillus stearothermophilus B-2194 thermostable purine-nucleoside phosphorylase II (EC 2.4.2.1). Use of the cells containing this thermostable enzyme allowed the process to be conducted at a temperature of 70 degrees C, which provided the maximal concentrations of sparingly soluble substrates. The best results were obtained with 2-chloroadenine as a modified base. The highest yield of the target 2-chloro-2'-deoxyadenosine (up to 95% in the case of deoxyguanosine) was reached when using 2'-deoxypurines as donors of deoxyribose. Use of thymidine for these purposes required its considerable molar excess over 2-chloroadenine (up to 6 : 1), which is connected with a nonoptimal amount of endogenous thymidine phosphorylase, necessary for synthesis of deoxyribose-1-phosphate, in the transglycosylation reaction. 相似文献
9.
Huang Y O'Mara B Conover M Ludwig R Fu J Tao L Li ZJ Rieble S Grace MJ Russell RJ 《Protein science : a publication of the Protein Society》2012,21(5):625-632
A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation. 相似文献
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【背景】D-甘露糖的酶促转化方法已受到相当大的关注。【目的】研究D-葡萄糖异构酶(D-glucoseisomerase,D-GIase)和D-来苏糖异构酶(D-lyxoseisomerase,D-LIase)共表达于大肠杆菌细胞生产D-甘露糖的工艺条件。【方法】将D-GIase和D-LIase基因片段合成后酶切连接到载体p CDFDuet-1上,构建p CDFDuet-Acce-DGI/Peba-DLI重组质粒并导入到大肠杆菌BL21(DE3)中共表达,通过摇瓶培养得到产D-GIase和D-LIase的菌体,测定该共表达细胞体系的反应条件。【结果】添加1 mmol/L Co~(2+),共表达体系酶的最适温度和p H分别为70°C和6.0。以浓度分别为100、300、500 g/L的D-葡萄糖为底物生产D-甘露糖,平衡后D-甘露糖质量浓度分别为13.8、38.1、62.6 g/L,相应的转化率分别为13.8%、12.7%、12.5%,D-葡萄糖、D-果糖和D-甘露糖的平衡比约为50:37.5:12.5。【结论】D-GIase和D-LIase在大肠杆菌细胞中组成的共表达体系通过一锅法可利用D-葡萄糖为底物生产D-甘露糖。 相似文献
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V A Efimov I V Aleksiuk A A Buriakova I N Pashkova N P Skiba O G Chakhmakhcheva 《Bioorganicheskaia khimiia》1989,15(8):1078-1090
Expression of the synthetic gene for human proinsulin in E. coli has been investigated. The proinsulin gene has been expressed directly under the control of a synthetic promoter of phage fd DNA and a promoter of tryptophan operon, or using fusions with fragments of some bacterial proteins. These fusions gave insoluble polypeptide products amounting to 20-30% of total cellular protein. The scheme for isolating proinsulin from bacterial cells was developed. Proinsulin was cleaved from leader polypeptides by treatment with cyanogen bromide and converted into human insulin. 相似文献
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Honghong J Fuping L Yu L Xiaoguang L Yihan L Hongbin W Jing L Yueting C 《Biotechnology letters》2011,33(6):1145-1150
Glucokinase (glk), phosphomannomutase (manB), and mannose-1-phosphate guanylytransferase (manC) are needed for the biosynthesis of GDP-mannose. A recombinant E. coli strain over-expressing these three genes was constructed to produce guanosine 5'-diphosphate (GDP)-mannose, the donor of GDP-fucose, an essential substrate for synthesis of fucosyloligosaccharides. In addition, the glk, manB, and manC genes were individually cloned into the expression vector pET-22b (+) to construct three recombinant E. coli strains pET-glk, pET-manB and pET-manC, respectively. Fermentation of the recombinant strain BL21/pET-glk-manB-manC had a conversion rate of 23% from mannose to GDP-mannose under IPTG induction, while coupling fermentation of the three recombinant strains BL21/pET-glk, BL21/pET-manB, BL21/pET-manC resulted in a conversion rate of 33% under the same induction conditions. 相似文献
13.
Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc. 相似文献
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Xiang-Yang Fu 《Applied microbiology and biotechnology》2010,88(1):75-86
Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant
proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane
remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture
medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the
cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins
could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The
tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins
in E. coli. 相似文献
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An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability. 相似文献
17.
Safina DR Rafieva LM Koval' AV Shkurina EE Dmitrieva VG Raevskaia NM Gasanov EV Demidiuk IV Kostrov SV 《Bioorganicheskaia khimiia》2008,34(3):327-332
Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3lambda) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure. 相似文献
18.
Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed. 相似文献
19.
Using the Escherichia coli OmpC protein as an anchoring motif, four different poly-His units (1, 2, 3 and 6 copies of 6-His) were displayed on the seventh loop of the OmpC. Recombinant E. coli strains displaying 1, 3 or 6 copies of poly-His became much more sensitive to SDS (0.1%, w/v) and EDTA (2 mM) compared with control strains. However, recombinant E. coli cells displaying 2 copies of poly-His were resistant to SDS and EDTA; greater than 70% and 90% of cells maintained cell integrity after 60 min treatment with SDS and EDTA, respectively, suggesting its usefulness as a whole cell biosorbent. 相似文献
20.
Si Jae Park Eun Young Kim Won Noh Young Hoon Oh Hye Young Kim Bong Keun Song Kwang Myung Cho Soon Ho Hong Seung Hwan Lee Jonggeon Jegal 《Bioprocess and biosystems engineering》2013,36(7):885-892
In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD600 of 2–10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD600 of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al2O3 as catalyst in toluene with the yield of 96 %. 相似文献