Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Cloning of full length or NT Δ-17 (ω-3) fatty acid desaturase. (A) The prediction of Δ-17 (ω-3) fatty acid desaturase transmembrane domain using TMpred ( http://www.ch.embnet.org/software/TMPRED_form.html ). (B) pPICZαA and Δ-17 (full length) or Δ-17 (NT) were generated using the pPICZαA expression vector and both digested with Xho I and Not I, and connected using T4 DNA ligase. (C) The relative plasmids were digested using the restriction enzyme Xba I and Xho I. NT, without transmembrane domain.

Article Snippet: Then the recombinant plasmids, Δ-17NT and Δ-17FL, and expression vector, pPICZαA, were linearized using the restriction enzyme SalI (Takara Bio, Inc.).

Techniques: Clone Assay, Software, Generated, Expressing, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Expression and optimization conversion efficiency of Δ-17NT or Δ-17 full length. (A) The linearization working model of pPICZαA, Δ-17NT or Δ-17 full length. (B) Preparation of Pichia pastoris competent cells. Polymerase chain reaction was used to detect the fragment of (C) Δ-17NT and (D) Δ-17 full length transfected into P. pastoris . NT, without transmembrane domain. Lanes 1–9: X-33/pPICZαA-Δ-17NT (Δ-17 FL) transformants; Lane 10: Vector pPICZαA-Δ-17NT (Δ-17 FL) as positive control; Lane 11: X-33 as negative control. AOX1, alcohol oxidase 1; CYC1, cytochrome c1.

Article Snippet: Then the recombinant plasmids, Δ-17NT and Δ-17FL, and expression vector, pPICZαA, were linearized using the restriction enzyme SalI (Takara Bio, Inc.).

Techniques: Expressing, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Positive Control, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: (A) Following culture in conditioned medium of pPICZαA-Δ-17FL with induction for 96 h at 28°C, the expression of Δ-17FL using different Pichia pastoris monoclones was measured by SDS-PAGE. BCA was used as a PC and conditioned medium of pPICZαA-Δ-17FL without induction was used as a NC. Lane 1, 3 and 4 demonstrated pPICZαA-Δ-17FL induction. (B) Similarly, the expression of Δ-17NT was measured using SDS-PAGE. FL, full length; NT, without transmembrane domain; PC, positive control; NC, negative control; BCA, bicinchoninic acid.

Article Snippet: Then the recombinant plasmids, Δ-17NT and Δ-17FL, and expression vector, pPICZαA, were linearized using the restriction enzyme SalI (Takara Bio, Inc.).

Techniques: Expressing, SDS Page, Positive Control, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Effect of pPICZαA-Δ-17NT expression on cell viability and apoptosis in vitro . (A) MTT assay was used to measure HepG2 cell viability, equal numbers of cells were transfected with pPICZαA-Δ-17NT or vector and cultured. MTT assay was performed every 24 h. Data are presented as the mean ± standard deviation. (B) DNA fragmentation terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. The apoptosis level was measured every 24 h. Data are presented as the mean + standard deviation. The fold change is relative to vector at 0 h. (C) The level of GSK-3β phosphorylation and β-catenin expression was demonstrated in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. (D) The level of cleavage of caspase-9 and caspase-3 was demonstrated in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. # P<0.001 and *P<0.05 vs. vector at the same time point. NT, without transmembrane domain; vector, control vector; OD, optical density.

Article Snippet: Then the recombinant plasmids, Δ-17NT and Δ-17FL, and expression vector, pPICZαA, were linearized using the restriction enzyme SalI (Takara Bio, Inc.).

Techniques: Expressing, In Vitro, MTT Assay, Transfection, Plasmid Preparation, Cell Culture, Standard Deviation, TUNEL Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Cloning of full length or NT Δ-17 (ω-3) fatty acid desaturase. (A) The prediction of Δ-17 (ω-3) fatty acid desaturase transmembrane domain using TMpred ( http://www.ch.embnet.org/software/TMPRED_form.html ). (B) pPICZαA and Δ-17 (full length) or Δ-17 (NT) were generated using the pPICZαA expression vector and both digested with Xho I and Not I, and connected using T4 DNA ligase. (C) The relative plasmids were digested using the restriction enzyme Xba I and Xho I. NT, without transmembrane domain.

Article Snippet: The expression vector, pPICZαA (an affiliate of Promega (Beijing) Biotech Co., Ltd, Beijing, China), was digested with XhoI and NotI. pMD™ 18-T Vector Cloning kit (Takara Bio, Inc.; catalogue no. 6011) was used to connect pPICZαA and Δ-17 (FL) or Δ-17 (NT) to obtain the recombinant plasmid, following the manufacturers protocol.

Techniques: Clone Assay, Software, Generated, Expressing, Plasmid Preparation

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Expression and optimization conversion efficiency of Δ-17NT or Δ-17 full length. (A) The linearization working model of pPICZαA, Δ-17NT or Δ-17 full length. (B) Preparation of Pichia pastoris competent cells. Polymerase chain reaction was used to detect the fragment of (C) Δ-17NT and (D) Δ-17 full length transfected into P. pastoris . NT, without transmembrane domain. Lanes 1–9: X-33/pPICZαA-Δ-17NT (Δ-17 FL) transformants; Lane 10: Vector pPICZαA-Δ-17NT (Δ-17 FL) as positive control; Lane 11: X-33 as negative control. AOX1, alcohol oxidase 1; CYC1, cytochrome c1.

Article Snippet: The expression vector, pPICZαA (an affiliate of Promega (Beijing) Biotech Co., Ltd, Beijing, China), was digested with XhoI and NotI. pMD™ 18-T Vector Cloning kit (Takara Bio, Inc.; catalogue no. 6011) was used to connect pPICZαA and Δ-17 (FL) or Δ-17 (NT) to obtain the recombinant plasmid, following the manufacturers protocol.

Techniques: Expressing, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Positive Control, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: (A) Following culture in conditioned medium of pPICZαA-Δ-17FL with induction for 96 h at 28°C, the expression of Δ-17FL using different Pichia pastoris monoclones was measured by SDS-PAGE. BCA was used as a PC and conditioned medium of pPICZαA-Δ-17FL without induction was used as a NC. Lane 1, 3 and 4 demonstrated pPICZαA-Δ-17FL induction. (B) Similarly, the expression of Δ-17NT was measured using SDS-PAGE. FL, full length; NT, without transmembrane domain; PC, positive control; NC, negative control; BCA, bicinchoninic acid.

Article Snippet: The expression vector, pPICZαA (an affiliate of Promega (Beijing) Biotech Co., Ltd, Beijing, China), was digested with XhoI and NotI. pMD™ 18-T Vector Cloning kit (Takara Bio, Inc.; catalogue no. 6011) was used to connect pPICZαA and Δ-17 (FL) or Δ-17 (NT) to obtain the recombinant plasmid, following the manufacturers protocol.

Techniques: Expressing, SDS Page, Positive Control, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Purification and function analysis of the Δ-17 fatty acid desaturase with or without transmembrane domain

doi: 10.3892/etm.2017.4790

Figure Lengend Snippet: Effect of pPICZαA-Δ-17NT expression on cell viability and apoptosis in vitro . (A) MTT assay was used to measure HepG2 cell viability, equal numbers of cells were transfected with pPICZαA-Δ-17NT or vector and cultured. MTT assay was performed every 24 h. Data are presented as the mean ± standard deviation. (B) DNA fragmentation terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. The apoptosis level was measured every 24 h. Data are presented as the mean + standard deviation. The fold change is relative to vector at 0 h. (C) The level of GSK-3β phosphorylation and β-catenin expression was demonstrated in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. (D) The level of cleavage of caspase-9 and caspase-3 was demonstrated in HepG2 cells transfected with pPICZαA-Δ-17NT or vector. # P<0.001 and *P<0.05 vs. vector at the same time point. NT, without transmembrane domain; vector, control vector; OD, optical density.

Article Snippet: The expression vector, pPICZαA (an affiliate of Promega (Beijing) Biotech Co., Ltd, Beijing, China), was digested with XhoI and NotI. pMD™ 18-T Vector Cloning kit (Takara Bio, Inc.; catalogue no. 6011) was used to connect pPICZαA and Δ-17 (FL) or Δ-17 (NT) to obtain the recombinant plasmid, following the manufacturers protocol.

Techniques: Expressing, In Vitro, MTT Assay, Transfection, Plasmid Preparation, Cell Culture, Standard Deviation, TUNEL Assay