scholarly journals An Alkane-Responsive Expression System for the Production of Fine Chemicals

1999 ◽  
Vol 65 (6) ◽  
pp. 2324-2332 ◽  
Author(s):  
Sven Panke ◽  
Andreas Meyer ◽  
Caroline M. Huber ◽  
Bernard Witholt ◽  
Marcel G. Wubbolts
Keyword(s):  
Specific Activity ◽  
Styrene Oxide ◽  
Expression System ◽  
Regulatory System ◽  
Dry Weight ◽  
Pseudomonas Oleovorans ◽  
Structural Genes ◽  
Biotechnological Processes ◽  
Terminal Amino

ABSTRACT Membrane-located monooxygenase systems, such as thePseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.

DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
M Ph Verbeet ◽  
R F Evers ◽  
A Leyte ◽  
H L Lamain ◽  
A J J Van Ooyen ◽  
...  
Keyword(s):  
Factor Viii ◽  
Recombinant Proteins ◽  
Cleavage Site ◽  
Specific Activity ◽  
Expression System ◽  
Full Length ◽  
Procoagulant Activity ◽  
Expression Vectors ◽  
Recombinant Fviii

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.

The use of p-Iodophenyl[125I] isothiocyanate for determination of N-terminal amino acids in nanomole quantities of protein

1975 ◽  
Vol 28 (10) ◽  
pp. 2289 ◽  
Author(s):  
CJ Burrell ◽  
PD Cooper ◽  
JM Swan
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coupling Reaction ◽  
Molar Content ◽  
Terminal Residue ◽  
Terminal Amino

Identification of the N-terminal residue of 2.5-5 nmol of protein was possible by means of a modified Edman reaction and p-iodophenyl[125I] isothiocyanate of high specific activity. The second and third residues could also be identified. Yields of the terminal residue were 25-50%, allowing approximate quantitation of the molar content of protein. Use of the isotope allowed certain steps of the degradative cycle to be examined in some detail, and preparation of this relatively non- volatile labelled Edman reagent was safe, cheap and convenient. ��� The synthesis and properties of the p-iodophenylthiohydantoins of 19 amino acids, and some side products of the coupling reaction, are described.

scholarly journals Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

2003 ◽  
Vol 69 (8) ◽  
pp. 4985-4988 ◽  
Author(s):  
Laurence Girbal ◽  
Isabelle Mortier-Barrière ◽  
Frédéric Raynaud ◽  
Céline Rouanet ◽  
Christian Croux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Acetobutylicum ◽  
Time Course ◽  
Specific Activity ◽  
Expression System ◽  
Regulatory System ◽  
Inducible Promoter ◽  
Reporter System ◽  
Inducible Gene

ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

scholarly journals Towards a Biocatalyst for (S)-Styrene Oxide Production: Characterization of the Styrene Degradation Pathway of Pseudomonas sp. Strain VLB120

1998 ◽  
Vol 64 (6) ◽  
pp. 2032-2043 ◽  
Author(s):  
Sven Panke ◽  
Bernard Witholt ◽  
Andreas Schmid ◽  
Marcel G. Wubbolts
Keyword(s):  
Kinetic Resolution ◽  
Response Regulator ◽  
Styrene Oxide ◽  
Enantiomeric Excess ◽  
Dry Weight ◽  
High Rate ◽  
Transcriptional Level ◽  
Structural Genes ◽  
Styrene Monooxygenase ◽  
Six Genes

ABSTRACT In order to design a biocatalyst for the production of optically pure styrene oxide, an important building block in organic synthesis, the metabolic pathway and molecular biology of styrene degradation inPseudomonas sp. strain VLB120 was investigated. A 5.7-kbXhoI fragment, which contained on the same strand of DNA six genes involved in styrene degradation, was isolated from a gene library of this organism in Escherichia coli by screening for indigo formation. T7 RNA polymerase expression experiments indicated that this fragment coded for at least five complete polypeptides, StyRABCD, corresponding to five of the six genes. The first two genes encoded the potential carboxy-terminal part of a sensor, named StySc, and the complete response regulator StyR. Fusion of the putative styAp promoter to a lacZreporter indicated that StySc and StyR together regulate expression of the structural genes at the transcriptional level. Expression ofstyScR also alleviated a block that prevented translation of styA mRNA when a heterologous promoter was used. The structural genes styA and styBproduced a styrene monooxygenase that converted styrene to styrene oxide, which was then converted to phenylacetaldehyde by StyC. Sequence homology analysis of StyD indicated a probable function as a phenylacetaldehyde dehydrogenase. To assess the usefulness of the enzymes for the production of enantiomerically pure styrene oxide, we investigated the enantiospecificities of the reactions involved. Kinetic resolution of racemic styrene oxide by styrene oxide isomerase was studied with E. coli recombinants carryingstyC, which converted styrene oxide at a very high rate but with only a slight preference for the S enantiomer. However, recombinants producing styrene monooxygenase catalyzed the formation of (S)-styrene oxide from inexpensive styrene with an excellent enantiomeric excess of more than 99% at rates up to 180 U g (dry weight) of cells−1.

An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

1983 ◽  
Vol 50 (03) ◽  
pp. 740-744 ◽  
Author(s):  
Nils Bergsdorf ◽  
Torbjörn Nilsson ◽  
Per Wallén
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Thromboembolic Disease ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

BIOASSAY OF PROLACTIN

1969 ◽  
Vol 60 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Charles S. Nicoll
Keyword(s):  
Epithelial Cells ◽  
Lipid Content ◽  
Dry Weight ◽  
Fat Content ◽  
Assay Sensitivity ◽  
Mucosal Epithelium ◽  
Wet Weight ◽  
Total Dry Weight

ABSTRACT The response of the pigeon crop-sac to systemically acting prolactin (injected subcutaneously) was evaluated by measuring the wet weight of the responsive lateral lobes of the organ and by determining the dry weight of a 4 cm diameter disc of mucosal epithelium taken from one hemicrop. Of several different injection schedules tested, administration of prolactin in four daily injections was found to yield optimal responses. When compared with a graded series of prolactin doses, measurement of the mucosal dry weight proved to be a better method of response quantification than determination of the crop-sac wet weight with respect to both assay sensitivity and precision. The submucosal tissue of the crop-sac was estimated to constitute about 64 % of the total dry weight of the unstimulated organ and it was found to be relatively unresponsive to prolactin stimulation in comparison with the mucosa. The lipid content of the mucosal epithelium was determined using unstimulated crop-sacs or tissues which showed varying degrees of prolactin-induced proliferation. The fat content of the mucosal epithelial cells increased only slightly more rapidly than the dry weight or the defatted dry weight of the mucosa. Suggestions are made for the further improvement of the systemic crop-sac assay for prolactin.

Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture

2020 ◽  
Vol 36 (3) ◽  
pp. 82-89
Author(s):  
O.V. Gromova ◽  
O.S. Durakova ◽  
S.V. Generalov ◽  
L.F. Livanova ◽  
O.A. Volokh
Keyword(s):  
Cell Culture ◽  
Vibrio Cholerae ◽  
Production Control ◽  
Specific Activity ◽  
Methodological Approach ◽  
Toxin Production ◽  
Submerged Cultivation ◽  
Vaccine Production ◽  
Antigenic Activity

Том 36(2020) №3 стр. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89А.В. Гаева1*, О.В. Громова1, О.С. Дуракова1, С.В. Генералов1, Л.Ф. Ливанова1, О.А. Волох1 Определение специфической активности компонентов холерной химической вакцины с использованием культуры клеток 1ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб»» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, Саратов 410005 *[email protected] Поступила - 2019-11-26; После доработки - 2020-03-16; Принята к публикации - 2020-05-15 Список литературы Описаны методы определения динамики продукции токсинов штаммом Vibrio cholerae 569B при глубинном культивировании в биореакторе и антигенной активности специфической фракции холерогена-анатоксина по анатоксинсвязыванию с использованием клеточных культур. Показана высокая степень соответствия результатов, полученных методами, применяемыми для контроля этапов производства холерной химической вакцины и рассмотренными в данной работе. Отмечено, что применение клеточной линии СНО-К1 наиболее перспективно для замены биомоделей на промежуточных этапах контроля активных компонентов холерной химической вакцины. Разработанный методический подход впервые предлагается использовать на этапах производства холерной бивалентной химической вакцины. культура клеток, Vibrio cholerae, холерная химическая вакцина, контроль производства, холера. Vol 36(2020) N 3 p. 82-89; DOI 10.21519/0234-2758-2020-36-3-82-89A.V. Gaeva1*, O.V. Gromova1, O.S. Durakova1, S.V. Generalov1, L.F. Livanova1, O.A. Volokh1 Determination of Specific Activity of Cholera Chemical Vaccine Components using Cell Culture 1Russian Research Anti-Plague Institute «Microbe» of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Saratov, 410005 *[email protected] Received - 26.11.2019; Accepted - 15.05.2020 References The methods has been described to determine the dynamics of toxin production by the Vibrio cholerae 569B strain during submerged cultivation in bioreactor and of the antigenic activity of specific choleragen anatoxin fraction by anatoxin binding levels using cell cultures. High degree of consistency was observed between the results obtained via the method under consideration and those obtained via control methods at different stages of cholera chemical vaccine production. It was shown that the CHO-K1 cell line is the most promising substitute for biomodels at the intermediate stages of control of active cholera chemical vaccine components. The developed methodological approach was first proposed for use at the stages of cholera chemical bivalent vaccine manufacturing. cell culture, Vibrio cholerae, cholera chemical vaccine, production control, cholera.

The Natural Radioactivity in Food: A Comparison Between Different Feeding Regimes

2019 ◽  
Vol 15 (5) ◽  
pp. 493-499 ◽  
Author(s):  
Francesco Caridi ◽  
Santina Marguccio ◽  
Alberto Belvedere ◽  
Maurizio D`Agostino ◽  
Giovanna Belmusto
Keyword(s):  
Vegetable Oils ◽  
Natural Radioactivity ◽  
Specific Activity ◽  
Mean Value ◽  
Vegetable Food ◽  
Fish Samples ◽  
Feeding Regimes ◽  
Italian Origin ◽  
Dose Levels

Background: In this article a comprehensive study was carried out for the determination of natural radioactivity in animal and vegetable food (meat, fish, milk and derivates, legumes, cereals and derivates, fruit, hortalizas, vegetables, vegetable oils) typical of different feeding regimes, for the age category higher than 17 years. Methods: A total of eighty-five samples of Italian origin, coming from large retailers during the years 2014, 2015 and 2016, were analyzed through HPGe gamma spectrometry. Results: The specific activity of 40K was investigated and its mean value was found to be: (106.3 ± 6.9) Bq/kg for bovine, swine and sheep meat; (116.5 ± 9.7) Bq/kg for fish; (52.9 ± 3.1) Bq/kg for milk and derivates; (271.9 ± 16.7) Bq/kg for legumes; (67.2 ± 4.7) Bq/kg for cereals and derivates; (52.7 ± 4.4) Bq/kg for fruit; (72.9 ± 5.6) Bq/kg for hortalizas; (83.9 ± 6.5) Bq/kg for vegetables; lower than the minimum detectable activity for vegetable oils. For animal food the highest mean 40K activity concentration was found in fish samples; for vegetable food the highest one was detected in legumes. Conclusion: The evaluation of dose levels due to the food ingestion typical of Mediterranean, Vegetarian and Vegan diets was performed. The annual effective dose was found to be 0.16 mSv/y, 0.41 mSv/y and 0.54 mSv/y, respectively.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

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