scholarly journals Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

2020 ◽  
Vol 23 (2) ◽  
pp. First
Author(s):  
Hân Khả Lê ◽  
Nghĩa Hiếu Nguyễn ◽  
Oanh Cao Kiều Nguyễn ◽  
Nhân Trí Nguyễn
Keyword(s):  
Biological Activity ◽  
Pichia Pastoris ◽  
Cell Line ◽  
Target Gene ◽  
Promoter Sequence ◽  
Multifunctional Protein ◽  
Multiple Copies ◽  
One Step ◽  
And Migration ◽  
Nih 3T3

Introduction: Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an important role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expression systems. Methods: The recombinant Pichia pastoris carrying the fgf-2 gene was cloned by using homologous cloning method. The recombinant strains were screened by PCR reactions using specific primers for the target gene and the AOX1 promoter sequence. Moreover, the copy number of the fgf-2 gene inserted into the P. pastoris genome was identified by semi-quantitative PCR method. The secreted rhFGF-2 protein was collected in the induced BMMY medium at a final methanol concentration of 0.5%, and purified by one-step heparin affinity chromatography. The quantity and biological activity of the purified protein were determined by competitive ELISA method and MTT assay on NIH-3T3 cell line, respectively. Results: Various recombinant P. pastoris clones carrying different copy numbers of the fgf-2 gene were obtained and categorized into 3 groups: the low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). Among those strains, the 4-copy one produced the rhFGF-2 protein at the highest expression level. After purification, the purity of rhFGF-2 protein reached 98.8%, and the recovery yield was 179.2 µg of protein from 200 mL of flask culture (equivalent to 850 µg/L). The purified rhFGF-2 protein showed similar biological activity on NIH-3T3 cell line with the commercial FGF-2 protein. Conclusion: The recombinant FGF-2 protein was successfully secretory expressed from Pichia pastoris, and successfully purified by only one-step chromatography.

Multiple copies of the target gene enhances plectasin secretion in Pichia pastoris X-33

2015 ◽  
Vol 50 (4) ◽  
pp. 553-560 ◽  
Author(s):  
Da Teng ◽  
Di Xi ◽  
Jun Zhang ◽  
Xiumin Wang ◽  
Ruoyu Mao ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Target Gene ◽  
Multiple Copies

scholarly journals Retroviral expression of alternatively spliced forms of rat fibronectin.

1990 ◽  
Vol 110 (3) ◽  
pp. 833-847 ◽  
Author(s):  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Cell Line ◽  
Cell Types ◽  
Retroviral Vectors ◽  
Biologically Active ◽  
Adherent Cell ◽  
Transformed Cell Line ◽  
Alternatively Spliced ◽  
And Migration ◽  
Nih 3T3

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.

The Anticoagulant Activity of 3.3’-(p-Alkylthio-Benzylidene)-Bis-4-Hydroxycoumarins and Their Oxidation Products

1967 ◽  
Vol 17 (01/02) ◽  
pp. 277-286 ◽  
Author(s):  
Maria Gumińska ◽  
M Eckstein ◽  
Barbara Stachurska ◽  
J Sulko
Keyword(s):  
Biological Activity ◽  
Anticoagulant Activity ◽  
Oxidation Products ◽  
Para Position ◽  
Sulphur Atom ◽  
Group I ◽  
One Step

SummaryThe anticoagulant activity of 3.3’-(benzylidene)-bis-4-hydroxycoumarin derivatives has been estimated by one step Quick’s method. The derivatives contained the following groups in the para position of benzylidene residue: NCS- (I), CH3-S- (II), CH3-SO-(III), CH3-S02- (IV), C2H5-S- (V), C2H5-SO- (VI), C2H5-S02- (VII). All these compounds were much more active than 3.3’-(benzylidene)-bis-4-hydroxycoumarin itself.Compounds possessing the ethyl chain at the sulphur atom (V, VI, VII) were more active than methyl homologues (II, III, IV). Comparison of the activity of the series of thio-, sulphoxy-, and sulphonyl-derivatives showed that among methyl- and ethyl-derivatives those with the sulphoxy grouping (III, VI) displayed the greatest anticoagulant activity. The action of sulphonyl (IV, VII) and thio-derivatives (II, V) was weaker and shortest. The derivative with the NCS-group (I) possessed a relatively the lowest activity among the investigated compounds. 3.3’-(p-Ethylsulphoxybenzyl-idene)-bis-4-hydroxycoumarin (VI), with distinct biological activity reached about ½ of dicoumarol activity.

Role of microenvironment on proliferation and migration of an SDHB silenced murine Pheochromocytoma cell line

2017 ◽  
Author(s):  
Serena Martinelli ◽  
Vanessa D'Antongiovanni ◽  
Susan Richter ◽  
Letizia Canu ◽  
Tonino Ercolino ◽  
...  
Keyword(s):  
Cell Line ◽  
Pheochromocytoma Cell ◽  
And Migration ◽  
Proliferation And Migration

Synthesis of New α-Amino Phosphonates Containing 3-Amino-4(3H) Quinazolinone Moiety as Anticancer and Antimicrobial Agents: DFT, NBO, and Vibrational Studies

2018 ◽  
Vol 15 (2) ◽  
pp. 286-296 ◽  
Author(s):  
Mohamed K. Awad ◽  
Mahmoud F. Abdel-Aal ◽  
Faten M. Atlam ◽  
Hend A. Hekal
Keyword(s):  
Biological Activity ◽  
Cell Line ◽  
Quantum Chemical ◽  
Density Functional ◽  
Carcinoma Cell ◽  
Liver Carcinoma ◽  
Anticancer Activities ◽  
Functional Theory ◽  
Ft Ir ◽  
Good Agreement

Aim and Objective: Synthesis of new .-aminophosphonates containing quinazoline moiety through Kabachnik-Fields reaction in the presence of copper triflate catalyst [32], followed by studying their antimicrobial activities and in vitro anticancer activities against liver carcinoma cell line (HepG2) with the hope that new anticancer agents could be developed. Also, the quantum chemical calculations are performed using density functional theory (DFT) to study the effect of the changes of molecular and electronic structures on the biological activity of the investigated compounds. Materials and Method: The structures of the synthesized compounds are confirmed by FT-IR, 1H NMR, 13C NMR, 31P NMR and MS spectral data. The synthesized compounds show significant antimicrobial and also remarkable cytotoxicity anticancer activities against liver carcinoma cell line (HepG2). Density functional theory (DFT) was performed to study the effect of the molecular and electronic structure changes on the biological activity. Results: It was found that the electronic structure of the substituents affects on the reaction yield. The electron withdrawing substituent, NO2 group 3b, on the aromatic aldehydes gave a good yield more than the electron donating substituent, OH group 3c. The electron deficient on the carbon atom of the aldehydic group may increase the interaction of the Lewis acid (Cu(OTf)2) and the Lewis base (imine nitrogen), and accordingly, facilitate the formation of imine easily, which is attacked by the nucleophilic phosphite species to give the α- aminophosphonates. Conclusion: The newly synthesized compounds exhibit a remarkable inhibition of the growth of Grampositive, Gram-negative bacteria and fungi at low concentrations. The cytotoxicity of the synthesized compounds showed a significant cytotoxicity against the liver cancer cell line (HepG 2). Also, it was shown from the quantum chemical calculations that the electron-withdrawing substituent increases the biological activity of the α-aminophosphonates more than the electron donating group which was in a good agreement with the experimental results. Also, a good agreement between the experimental FT-IR and the calculated one was found.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Mariko Suzuki ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
...  
Keyword(s):  
Target Gene ◽  
Animal Experiments ◽  
Adenovirus Vector ◽  
Mouse Embryonic Fibroblast ◽  
Newborn Mouse ◽  
Guide Rnas ◽  
One Step ◽  
Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

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