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41.
采用优化组合保护剂冷冻干燥20株极端嗜盐菌,于4℃冷库保存2年,经复水测定,全部菌株保持较高存活性.用TENS法对其中9株菌R1,RF1,J7,S9,T4~1,J6,R6~1,R6~7和R6~5作质粒稳定性检测,含质粒的菌株RF1,J7,S9,T4~1,R6~5于4℃保存2年后,质粒稳定存在;而菌株R1,J8,R6~1和R6~7冻干保存前后均末检测到质粒.结果表明:优化组合保护剂不仅能有效地用于极端嗜盐菌的冷冻干燥保藏,还能保持宿主质粒的稳定性. 相似文献
42.
N. L. C. Licinio Da Silva A. A. Salgueiro W. M. Ledingham E. H. M. Melo J. L. Lima Filho 《Applied biochemistry and biotechnology》1992,37(1):1-10
The influence of potassium on ethanol production bySaccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated. This plasmid carries the glutamate dehydrogenase gene
conferring an 11-fold higher level of expressed enzyme activity over the wild type cells. All experiments were carried out
in batch culture with medium supplemented to different potassium concentrations up to 180 mM. Maximum ethanol production rate
was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions. Glucose uptake rate increased with
increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains. Furthermore, the
wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells. These lower
rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater
energy requirements by these cells due the presence of the plasmid. 相似文献
43.
Carole Rais-Beghdadi Mario A. Roggero Nicolas Fasel Christophe D. Reymond 《Applied biochemistry and biotechnology》1998,74(2):95-103
The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure
and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and
result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the
preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale
purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag
and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag
inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous
amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced
without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative
to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins. 相似文献
44.
Qinying Lin Joseph K. -K. Li Hing-Yat Peter Lam 《Applied biochemistry and biotechnology》1997,66(3):269-279
Using the crossover-linker mutagenesis method, the 5’ noncoding region of the λML-1 cDNA, which encodes the ligninase H8 isozyme
of the white-rot fungus,Phanerochaete chrysosporium, was deleted with the simultaneous insertion of the putativeSpodoptera frugiperda ribosome-binding sequence (RBS) (TATAAAT) directly in front of the translation-initiation codon of this gene. A recombinant
baculovirus, pVL-Mu-H8, carrying the ligninase-H8 gene was successfully constructed, as determined by both sequence analysis
and dot blot hybridization. A more than 18-fold increase in the expression of ligninase H8, compared to the previous pEV11-1A.3
recombinant baculovirus, was detected in the Sf-21 insect cells. This enzyme was detected within 3 d postinfection and was
biologically active, capable of oxidizing the model lignin compound, veratryl alcohol. The molecular weight of the overexpressed
42 kD protein was similar to that of the native fungal ligninase-H8 isozyme and it also reacted specifically with the anti-H8
monoclonal antibody (MAb 2D4.9) in Western blot analysis. 相似文献
45.
46.
A maize genomic clone containing a zein gene (Z4) is inserted into the T-DNA of the Ti plasmid pTiT37. Agrobacterium tumefaciens strain harboring this modified Ti plasmid is used to infect stem sections of young plants or explants of dicotyledonous Solanum nigrum. Axenic transformed calli active in nopaline synthesis are obtained and transgenic plants are differentiated from them DNA Southern hybridization and RNA dot-hybridization analyses show that the zein gene is really transferred and integrated into the nuclear genome of transformed Solanum nigrum and that the zein gene can be transcribed into mRNA in the transformed calli and shoots. But the presence of the zein protein cannot be detected in either the transformed calli or the transgenic shoots. The results of thte experiments demonstrate that the promoter of a gene from monocotyledonous plants can function normally in transgenic dicots. The possibility of developmentally-regulated expression of the zein gene in transformed dicots is discussed in 相似文献
47.
Recombinant antibodies for environmental analysis 总被引:1,自引:0,他引:1
Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples. 相似文献
48.
Gian Maria D'Amici Anna Maria Timperio Federica Gevi Giuliano Grazzini Lello Zolla 《Electrophoresis》2010,31(16):2730-2739
Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full‐length cDNA sequence of human factor VIII (FLrFVIII) or B‐domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate®, Helixate NexGen® and Refacto®), either FLrFVIII or BDDrFVIII, were investigated by 1‐ and 2‐DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2‐D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B‐domain‐truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGen® showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refacto® showed glutathione S‐transferase and β‐lactamase, whereas Advate® apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2‐DE is mandatory for revealing the presence of contaminants. 相似文献
49.
S. Irem Kaya Ahmet Cetinkaya Mehmet G. Caglayan Sibel A. Ozkan 《Electrophoresis》2022,43(9-10):1035-1049
Biopharmaceuticals (recombinant technology-based products, vaccines, whole blood and blood components, gene therapy, cells, tissues, etc.,) are described as biological medical products produced from various living sources such as human, microbial, animal, and so on by manufacturing, extraction, or semi-synthesis. They are complex molecules having high molecular weights. For their safety and efficacy, their structural, clinical, physicochemical, and chemical features must be carefully controlled, and they must be well characterized by analytical techniques before the approval of the final product. Capillary electrophoresis (CE) having versatile modes can provide valuable safety and efficacy information, such as amino acid sequence, size variants (low and high molecular weight variants), charged variants (acidic and basic impurities), aggregates, N-linked glycosylation, and O-linked glycosylation. There are numerous applications of CE in the literature. In this review, the most significant and recent studies on the analysis of recombinant DNA technology-based products using different CE modes in the last ten years have been overviewed. It was seen that the researches mostly focus on the analysis of mAbs and IgG. In addition, in recent years, researchers have started to prefer CE combined mass spectrometry (MS) techniques to provide a more detailed characterization for protein and peptide fragments. 相似文献
50.
Fanny Kieken Gaud Pinel Jean-Philippe Antignac Fabrice Monteau Anne Christelle Paris Marie-Agnès Popot Yves Bonnaire Bruno Le Bizec 《Analytical and bioanalytical chemistry》2009,394(8):2119-2128
Despite the worldwide existing regulation banning the use of the recombinant equine growth hormone (reGH) as growth promoter,
it is suspected to be used in horseracing to improve performances. Various analytical methods previously developed to screen
for its misuse have encountered some limitations in terms of detection timeframes, in particular during the first days following
reGH administration. A novel strategy involving the characterization of global metabolomic fingerprints in urine samples of
non-treated and reGH-treated horses by liquid chromatography–electrospray–high-resolution mass spectrometry (LC-ESI-HRMS)
is described and assessed in this paper in order to develop a new screening tool for growth hormone abuse in horseracing.
The strategy involves a limited sample preparation of the urine samples and the use of appropriate software for data processing
and analysis. As preliminary work, reproducibility of both sample preparation and mass spectrometry (MS) measurements was
evaluated in order to demonstrate the reliability of the method. Application of the developed protocol on two horses demonstrated
the suitability of the developed strategy and preliminary results showed significant modifications of the metabolome after
treatment with reGH.
相似文献