scholarly journals L-ARGININE AND L-GLUTAMIC ACID INCREASE THE CONTENT OF PROTEIN C IN THE EARLY STAGES OF ISOLATION FROM DONOR PLASMA

2021 ◽  
Vol 14 (3) ◽  
pp. 30-38
Author(s):  
I. I. Patalakh ◽  
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Protein C ◽  
Large Scale ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Scale Production ◽  
Amidolytic Activity ◽  
Initial Dilution ◽  
Purification Stage

Current large-scale production of blood-derived pharmacological preparations is aimed at expanding the list of products and deeper extraction of target proteins especially at the pre-purification stage. In particular, this problem becomes critical for the isolation of proteins like protein C (PC), which is present in plasma in trace amounts. Aim. We aimed to improve the buffer composition to minimize the interaction of PC with other proteins and lipids that are inevitably present in the stock material. Methods. The content of protein C in plasma and its derivatives was assessed by the amidolytic activity to the chromogenic substrate S2366. A decrease in homologous impurities and plasma enrichment with protein C was provided by selective bulk adsorption on DEAE-cellulose. Results. Here we describe that an equimolar mixture of two amino acids (L-arginine and L-glutamic acid) essentially increased the content of protein C at the stage of cryo-depleted plasma pre-purification, including initial dilution and subsequent enrichment of plasma with protein C due to selective bulk adsorption on DEAE- cellulose. Additionally, it was revealed that solutions of these amino acids, when combined, inhibit the induced amidolytic activity of protein C and increase its solubility (in contrast to other plasma proteases). Conclusion. Pre-adding of a mixture of amino acids L-arginine and L-glutamic acid to cryo-depleted plasma significantly optimizes the pre-purification stage of protein C, providing a 5-fold increase in its yield after elution from DEAE-cellulose.

Large-Scale Production and Properties of Immunoaffinity- Purified Human Activated Protein C Concentrate

Vox Sanguinis ◽  
1995 ◽  
Vol 69 (4) ◽  
pp. 309-318
Author(s):  
Carolyn L. Orthner ◽  
Annemarie H. Ralston ◽  
Dan Gee ◽  
Randy Kent ◽  
Billy Kolen ◽  
...  
Keyword(s):  
Protein C ◽  
Large Scale ◽  
Activated Protein C ◽  
Scale Production ◽  
Large Scale Production ◽  
Protein C Concentrate

scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

scholarly journals 300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

2014 ◽  
Vol 80 (13) ◽  
pp. 4003-4011 ◽  
Author(s):  
Colin J. Jackson ◽  
Christopher W. Coppin ◽  
Paul D. Carr ◽  
Alexey Aleksandrov ◽  
Matthew Wilding ◽  
...  
Keyword(s):  
Soluble Protein ◽  
Metal Ion ◽  
Large Scale ◽  
Computational Simulation ◽  
Active Form ◽  
Fold Increase ◽  
Soluble Form ◽  
Scale Production ◽  
Content Type ◽  
The Stability

ABSTRACTMicrobial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme fromArthrobacter aurescenswith environmental applications that catalyzes the hydrolysis of triazine herbicides, inEscherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

Large-Scale Production and Properties of Immunoaffinity-Purified Human Activated Protein C Concentrate

Vox Sanguinis ◽  
1995 ◽  
Vol 69 (4) ◽  
pp. 309-318 ◽  
Author(s):  
Carolyn L. Orthner ◽  
Annemarie H. Ralston ◽  
Dan Gee ◽  
Randy Kent ◽  
Billy Kolen ◽  
...  
Keyword(s):  
Protein C ◽  
Large Scale ◽  
Activated Protein C ◽  
Scale Production ◽  
Large Scale Production ◽  
Protein C Concentrate

scholarly journals Collagen peptide provides Streptomyces coelicolor CGMCC 4.7172 with abundant precursors for enhancing undecylprodigiosin production

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Xia Li ◽  
Meifeng Li ◽  
Junling Guo ◽  
Xian Liu ◽  
Xuepin Liao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Large Scale ◽  
Streptomyces Coelicolor ◽  
Malt Extract ◽  
Scale Production ◽  
Collagen Peptide ◽  
Amino Acid Availability ◽  
Large Scale Production ◽  
The Media

Abstract Effective and ecofriendly converting biomass to chemicals is important for sustainable engineering based on the foreseeable shortage of fossil resources. Undecylprodigiosin (UP) is a promising antibiotic, but the direct feeding of pure precursor amino acids makes it costly for large-scale production. Here, collagen peptide (CP), a renewable animal-derived biomass contains abundant precursor amino acids of UP. CP can act as carbon and nitrogen source for the growth of Streptomyces coelicolor CGMCC 4.7172. The plant biomasses including soybean meal, wheat bran, and malt extract were unsuitable for UP prodution. However, 365.40 µg/L UP was detected after 24 h in the media containing CP, and its highest concentration reached 1198.01 µg/L. UP was also detected in the media containing meat hydrolysates of domestic animals, but its initial production time was delayed, and final concentration was lower than that in the medium containing CP only. Compared the fermentation performances of CP and other proteins, CP has a special superiority for UP production. These results revealed that UP biosynthesis may be dependent on amino acid availability of substrates and CP is beneficial for UP production because of its specific amino acid composition. Graphical abstract

scholarly journals Highly Branched Polymers Based on Poly(amino acid)s for Biomedical Application

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1119
Author(s):  
Marisa Thompson ◽  
Carmen Scholz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Biomedical Applications ◽  
Large Scale ◽  
Biomedical Application ◽  
Building Blocks ◽  
Delivery Systems ◽  
Branched Polymers ◽  
Viral Gene

Polymers consisting of amino acid building blocks continue to receive consideration for biomedical applications. Since poly(amino acid)s are built from natural amino acids, the same building blocks proteins are made of, they are biocompatible, biodegradable and their degradation products are metabolizable. Some amino acids display a unique asymmetrical AB2 structure, which facilitates their ability to form branched structures. This review compares the three forms of highly branched polymeric structures: structurally highly organized dendrimers, dendrigrafts and the less organized, but readily synthesizable hyperbranched polymers. Their syntheses are reviewed and compared, methods of synthesis modulations are considered and variations on their traditional syntheses are shown. The potential use of highly branched polymers in the realm of biomedical applications is discussed, specifically their applications as delivery vehicles for genes and drugs and their use as antiviral compounds. Of the twenty essential amino acids, L-lysine, L-glutamic acid, and L-aspartic acid are asymmetrical AB2 molecules, but the bulk of the research into highly branched poly(amino acid)s has focused on the polycationic poly(L-lysine) with a lesser extent on poly(L-glutamic acid). Hence, the majority of potential applications lies in delivery systems for nucleic acids and this review examines and compares how these three types of highly branched polymers function as non-viral gene delivery vectors. When considering drug delivery systems, the small size of these highly branched polymers is advantageous for the delivery of inhalable drug. Even though highly branched polymers, in particular dendrimers, have been studied for more than 40 years for the delivery of genes and drugs, they have not translated in large scale into the clinic except for promising antiviral applications that have been commercialized.

scholarly journals Enhanced Plantlet Regeneration in Two Cacao (Theobroma cacao) Clones from Immature Inflorescence Explants

HortScience ◽  
2017 ◽  
Vol 52 (6) ◽  
pp. 892-895
Author(s):  
Jane Kahia ◽  
Siaka Kone ◽  
Lucien Diby ◽  
Georges Ngoran ◽  
Colombe Dadjo ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Survival Rate ◽  
Theobroma Cacao ◽  
Large Scale ◽  
Fold Increase ◽  
Plantlet Regeneration ◽  
Scale Production ◽  
Immature Inflorescence ◽  
Embryo Recovery ◽  
Planting Materials

Theobroma cacao L. (cacao) is a major tropical crop, grown for its oil-rich seed, which is used in the manufacture of chocolate, its derivatives, and cosmetics. Cacao is cultivated mainly by smallholders and represents a significant export commodity for some developing countries such as Côte d’Ivoire. It is conventionally propagated by seeds, grafting, and cuttings. Somatic embryogenesis offers an alternative method for propagation where large-scale production of planting materials is possible. In the current study, the effect of different concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin on induction of somatic embryogenesis and plantlet regeneration in two cocoa clones (coded as C1 and C14) were evaluated. Flowers were collected early in the morning, sterilized, explants excised and cultured on Driver, and Kuniyuki Walnut (DKW) media supplemented with different concentrations of 2, 4-D (9, 10, and 20 µM) and kinetin (0.5, 1, 2.5, 5, 10, and 25 µM) in separate experiments. The frequently used media in somatic embryogenesis of cacao [DKW supplemented with 0.022 µM thidiazuron (TDZ) and 9 µM 2, 4-D] was used as a control. The results of the study showed that explants cultured on media supplemented with 10 µM 2, 4-D and 5 µM kinetin produced the highest (28.0 ± 1.1) mean number of embryos/explant in C1 and this was a 9-fold increase in the number of embryos compared with the control. Explants cultured on media supplemented with 10 µM 2, 4-D and 2.5 µM kinetin produced the highest (7.0 ± 4.0) mean number of embryos/explant in C14 whereas the explants cultured on media supplemented with 20 µM 2, 4-D and 2.5 µM kinetin gave the highest (22.0 ± 1.7) mean number of embryos in clone C1 and C14. The regenerated embryos were germinated and successfully weaned in the green house with a survival rate of 70% being recorded. The paper describes an improved protocol compared with previous work in terms of embryo recovery and survival rate of the elite clones of cocoa through somatic embryogenesis. The results of the current study confirm that somatic embryogenesis of cacao clones is genotype dependent.

Purification and Characterization of Activation Fragments F1 and F2 from Human Prothrombin

1975 ◽  
Author(s):  
A. P. Ball ◽  
J. Fenton ◽  
D. L. Aronson ◽  
R. B. Franza ◽  
A. M. Young
Keyword(s):  
Gel Electrophoresis ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Scale Production ◽  
Purification And Characterization ◽  
Large Scale Production ◽  
Human Thrombin ◽  
A Chain

Considerable quantities of the non-thrombin portions of human prothrombin (II) have become available as a byproduct of the large-scale production of human thrombin (Biochim. Biophys. Acta 229, 26). Components not adsorbed on CG-50 are further purified by DEAE-cellulose chromatography and gel filtration, yielding the NH2-terminal fragment (F1) and the inner fragment (F2) which are homogeneous by SDS gel electrophoresis. SDS gel electrophoresis of reduced F1 indicates variable amounts of a two-chain derivative, F1’, with one chain migrating just ahead of F1 and one just ahead of the thrombin A-chain. The F1 → F1’ conversion is catalyzed by thrombin with the creation of a new MH2-terminal threonine. Ultracentrifugal patterns of human F1 and F2 closely resemble those of the bovine fragments. NH2-terminal residues were found to be alanine (± threonine) for F1 and serine for F2. Minor deviations from the reported amino acid compositions of bovine F1 and F2 were observed, primarily in the acidic residues. Other properties include:Immunization of rabbits with F1 gave a precipitating antibody to F1 which cross-reacts with II, but native F2 does not appear to be immunogenic. 3H-F1 is rapidly cleared from the blood of rabbits (T 1/2 20 min), with a major portion detectable in the urine.

scholarly journals New lipase-producing Streptomyces isolated from halo-alkaline habitat in Wadi El Natrun: polyphasic identification and statistical optimization of enzyme production

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohamed A. Mohamed ◽  
Hassan M. Awad
Keyword(s):  
Large Scale ◽  
Linseed Oil ◽  
Tween 80 ◽  
Fold Increase ◽  
Lipase Production ◽  
Medium Composition ◽  
Scale Production ◽  
Wide Ph Range ◽  
Wadi El Natrun ◽  
Ph Range

Abstract Background Bioprospecting lipase producers in non-conventional habitats are the way to find special enzymes of diverse applications. Halo-alkaline marshes in Wadi El Natrun in Egypt are some of the most stable ecological systems in the world, and because of the double extremities of alkalinity and salinity, they harbor individual microbes capable of adapting stress conditions. Results Eight strains were recovered from the coastline soil of Al-Beida Lake in Wadi El Natrun and have been tested for lipase production. Among the eight isolates, the strain SBLWN_MH2 was the most active producer of lipase (7.5 U/ml). The crude SBLWN_MH2 lipase showed activity over a wide pH range (3.5 to 13) with an optimum pH at 10.5, and it was able to show more than 75% of its highest activity at pH elevated up to 13. The identification using phenotypic and genotypic methods strongly indicated that the strain SBLWN_MH2 belonged to the genus Streptomyces with a similarity of 99%. Thus, it has been given the suggested name Streptomyces sp. SBLWN_MH2 (MG593538). SBLWN_MH2 produced extracellular lipase in modified starch casein medium supplemented with different oils or Tween-80, and the potential production rate has been attained in the case of linseed oil after 3 days. Further experiments have been carried out to optimize medium composition through Box-Behnken design and response surface methodology, and it was possible to achieve more than 3.5-fold increase in lipase production. Conclusions The present study indicates that Streptomyces sp. SBLWN_MH2 is a potential lipase producer and could be fruitfully employed in the large-scale production of highly alkaline lipase.

Advances in pollination techniques for large-scale seed production in Eucalyptusglobulus

2004 ◽  
Vol 52 (6) ◽  
pp. 781 ◽  
Author(s):  
Briony Patterson ◽  
Peter Gore ◽  
Brad M. Potts ◽  
René E. Vaillancourt
Keyword(s):  
Eucalyptus Globulus ◽  
Large Scale ◽  
Fold Increase ◽  
Microsatellite Analysis ◽  
Scale Production ◽  
Open Pollination ◽  
Weekly Cycle ◽  
Large Scale Production ◽  
Sib Families ◽  
Additive Genetic Effects

The effectiveness of supplementary pollination techniques for large-scale production of elite Eucalyptus globulus seed was tested by using pollen fixed for a rare allozyme. We assayed the paternity of 1954 seedlings derived from 1089 flowers on 14 trees with no emasculation or style isolation. We first compared the success of pollinating the transversely cut style surface with direct stigma pollination. For flowers pollinated up to 6 days after operculum shed, style cutting resulted in a 15-fold increase in the number of seed with the marker allozyme (intended pollination) compared with stigma pollination. There was no difference between either pollination treatment and open pollination in 7–14-day-old flowers. We subsequently pollinated 13 trees using the cut-style technique that resulted in 86.9% of progeny having the marker allozyme. Microsatellite analysis showed that only 4.8% of the progeny were selfs. Thus, we show that it is possible to obtain seed with low level of contamination and little selfing, without the costly steps of flower emasculation, isolation and labelling, by simply cutting the style just before pollination and pollinating all suitable flowers on a tree on a weekly cycle. By this approach, elite full-sib families can be generated for deployment into plantations to exploit both additive and non-additive genetic effects.

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