scholarly journals Efficient Production of Human Hyaluronidase PH20 in HEK293T Cells Using Site Specific Integration

2021 ◽  
Author(s):  
Nafiseh Sanei Ata-abadi ◽  
Kianoush Dormiani ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mahboube Forouzanfar ◽  
Leila Pirjamali ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Tumor Tissue ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Efficient Production ◽  
Phic31 Integrase ◽  
Cumulus Oocyte Complex ◽  
Site Specific Integration ◽  
Subcutaneous Space ◽  
Turbidimetric Assay

Abstract PH20 is hyaluronidase that hydrolyze the glycosidic bond of hyaluronic acid as a major proteoglycan found in extracellular matrices. PH20 is used in the subcutaneous space to increase the dispersion and absorption of co-administered drugs. PH20 is also injected against solid tumors for to better penetration of anticancer agents into the tumor tissue and inhibiting the tumor cell growth. In the present study, we have developed HEK293T stable cell lines secreting His-tagged human recombinant PH20 (rhPH20) in the culture supernatant through the PhiC31 integrase system. The produced rhPH20 was quantify using ELISA and turbidimetric assay and its activity was assessed through treatment of mouse cumulus-oocyte-complex (COCs). Furthermore, we have characterized genomic integration of PH20-containing vectors in one of the isolated clone with the highest levels of rhPH20 production. Our results demonstrated that the secreted rhPH20 in the culture supernatant contained a specific activity of approximately 3.5 IU/ml and it was properly able to denote all mouse oocytes. Consequently, it was revealed that PH20-expressing vectors integrated site-specifically in the PhiC31 pseudo attP sites in the host genome. Taken together, these results confirmed successful application of PhiC31 integrase as a robust approach for production of soluble, active rhPH20 in HEK293T cells.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

Screening and characterization of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 / Bacillus amyloliquefaciens EGE-B-2d.1’den termostabil fibrinolitik enzimin taranması ve karakterizasyonu

2016 ◽  
Vol 41 (3) ◽  
Author(s):  
Birkan Slem ◽  
Yüksel Gezgin ◽  
Rengin Eltem
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Fibrinolytic Activity ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Fibrinolytic Therapy ◽  
Fibrinolytic Enzyme ◽  
Fibrinolytic Enzymes ◽  
Enzyme Thermostability ◽  
Natural Agent

AbstractObjective: To screen fibrinolytic enzyme-producing Bacillus isolates (n=210) and to characterize of thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 that had the highest level of fibrinolytic activity together with the highest thermostability.Methods: Firstly, a total of 210 isolates were screened for their fibrinolytic enzyme production. The potent fibrinolytic enzyme producing isolates were evaluated for the thermostability of their fibrinolytic enzymes and one isolate showing prominent fibrinolytic activity was identified as molecular. Fermentation process was carried out on the isolate that had both the highest level of fibrinolytic activity and enzyme thermostability. The thermostable fibrinolytic enzyme from this isolate was then purified and characterized.Results: The fibrinolytic enzyme activities of 21 Bacillus sp. isolates in Nutrient Yeast Salt Medium were found to be in the range of 0.176-1.734 U/ml. The fibrinolytic activity of the enzyme purified from the culture supernatant of Bacillus amyloliquefaciens EGE-B-2d.1 was relatively stable at pH 7.0-11.0 for 24 h and also showed stability at a temperature of 60°C for 60 min. The enzyme degraded the fibrin clots by direct fibrinolysis. The specific activity and the molecular weight of the purified enzyme were estimated to be 44.46 units/mg protein and 30 kD respectively.Conclusion: The thermostable fibrinolytic enzyme from Bacillus amyloliquefaciens EGE-B-2d.1 was purified and characterized. This enzyme might also be used as a natural agent for oral fibrinolytic therapy or thrombosis prevention.

scholarly journals EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

2015 ◽  
Vol 2 (1) ◽  
pp. 25 ◽  
Author(s):  
Sri Sumarsih
Keyword(s):  
Culture Medium ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Agar Medium ◽  
Nitrilotriacetic Acid ◽  
Protoplast Transformation ◽  
E Coli ◽  
Recombinant Cells ◽  
Relative Molecular Weight ◽  
Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103127
Author(s):  
Edward A. Greenfield
Keyword(s):  
Culture Supernatant ◽  
Detection Method ◽  
Specific Activity ◽  
Binding Capacity ◽  
Sandwich Elisa ◽  
High Specific Activity ◽  
Tissue Culture Supernatant ◽  
Capture Assay ◽  
Antigen Capture ◽  
Elisa Plate

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

scholarly journals Studies on the Production of Fungal Peroxidases inAspergillus niger

2000 ◽  
Vol 66 (7) ◽  
pp. 3016-3023 ◽  
Author(s):  
Ana Conesa ◽  
Cees A. M. J. J. van den Hondel ◽  
Peter J. Punt
Keyword(s):  
Lignin Peroxidase ◽  
Manganese Peroxidase ◽  
Culture Medium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limiting Factors ◽  
Efficient Production ◽  
Mrna Levels ◽  
Extracellular Medium

ABSTRACT To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.

scholarly journals Altered Gingipain Maturation in vimA- and vimE-Defective Isogenic Mutants of Porphyromonas gingivalis

2005 ◽  
Vol 73 (3) ◽  
pp. 1357-1366 ◽  
Author(s):  
Elaine Vanterpool ◽  
Francis Roy ◽  
Lawrence Sandberg ◽  
Hansel M. Fletcher
Keyword(s):  
Cell Surface ◽  
Porphyromonas Gingivalis ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Phenotypic Effect ◽  
Western Blots ◽  
Defective Mutant ◽  
Surface Polysaccharide ◽  
Isogenic Mutants ◽  
Specific Sugar

ABSTRACT We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.

scholarly journals Purification of Pediocin PA-1 by Immunoaffinity Chromatography

2008 ◽  
Vol 91 (4) ◽  
pp. 828-832 ◽  
Author(s):  
Karim Naghmouchi ◽  
Djamel Drider ◽  
Ismail Fliss
Keyword(s):  
Cyanogen Bromide ◽  
Culture Supernatant ◽  
Specific Activity ◽  
Polyclonal Antibodies ◽  
Immunoaffinity Chromatography ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pediococcus Acidilactici ◽  
Purification Procedure ◽  
Average Recovery ◽  
One Step

Abstract A one-step purification procedure for purifying pediocin PA-1, a class IIa bacteriocin produced by Pediococcus acidilactici UL5, was developed based on column immunoaffinity chromatography with specific antipediocin PA-1 polyclonal antibodies coupled to cyanogen bromide-activated Sepharose. About 13.3 g/mL purified pediocin PA-1 was obtained from 15 mL P. acidilactici UL5 culture supernatant, as measured by enzyme-linked immunosorbent assay. The specific activity and average recovery of the eluted pediocin PA-1 were about 6602 AU/mg and 53.3, respectively. This is the first report of successful purification of pediocin PA-1 by immunoaffinity using pediocin PA-1-specific polyclonal antibodies.

scholarly journals Tumor microenvironment in focus: LA-ICP-MS bioimaging of a preclinical tumor model upon treatment with platinum(iv)-based anticancer agents

Metallomics ◽  
2015 ◽  
Vol 7 (8) ◽  
pp. 1256-1264 ◽  
Author(s):  
Sarah Theiner ◽  
Christoph Kornauth ◽  
Hristo P. Varbanov ◽  
Markus Galanski ◽  
Sushilla Van Schoonhoven ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Anticancer Agents ◽  
Tumor Tissue ◽  
Tumor Model ◽  
High Concentration ◽  
Concentration Gradients ◽  
Icp Ms

Bioimaging of Pt in tumor tissue exhibited unexpected high concentration gradients, correlating with histologic features.

Can carboplatin replace cisplatin for intraperitoneal use?

2008 ◽  
Vol 18 (Suppl 1) ◽  
pp. 29-32 ◽  
Author(s):  
K. Fujiwara
Keyword(s):  
Anticancer Agents ◽  
Large Scale ◽  
Randomized Trials ◽  
Tumor Tissue ◽  
Review Article ◽  
High Concentration ◽  
Peritoneal Surface ◽  
Platinum Agent ◽  
The Us ◽  
Future Potential

lntraperitoneal (IP) chemotherapy is theoretically a feasible route for treating ovarian cancer. It is possible to expose tumor tissue disseminated peritoneal surface to extremely high concentration of anticancer agents. Three large-scale, randomized trials conducted in the US have demonstrated a significant improvement of progression survival and/or overall survival in IP chemotherapy arm over intravenous arm. Despite these favorable results, IP chemotherapy has not been accepted as standard care. One of the reasons for this is the use of cisplatin, which has been replaced by the less toxic platinum agent, carboplatin, when administered intravenously. In this review article, we discuss why IP chemotherapy using carboplatin has been ignored and its future potential

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