Research ArticleResearch Article

Expression and Characterization of the Recombinant Human FLT-3 Ligand Extracellular Domain in Pichia Pastoris

Zongtang Huang and Xishan Hao
Chinese Journal of Clinical Oncology December 2006, 3 (6) 400-407;

Article Figures & Data

Figures

  • Synthesized cDNA of the FLT-3 ligand extracellular domain.
    Fig. 1.

    Synthesized cDNA of the FLT-3 ligand extracellular domain.

  • Construction of the expression vector.
    Fig. 2.

    Construction of the expression vector.

  • PCR analysis of positive anti-G418 yeast strain. Lane 1: KM71 strain DNA using 5’FL+3’FL primer as the negtive control; Lane 2: Positive strain DNA using 5’FL+3’AOX1 primer; Lane 3: Positive strain DNA using 5’AOX+ 3’FL primer; Lane 4: Positive strain DNA using 5’FL+3’FL;M: 100 bp ladder.
    Fig. 3.

    PCR analysis of positive anti-G418 yeast strain. Lane 1: KM71 strain DNA using 5’FL+3’FL primer as the negtive control; Lane 2: Positive strain DNA using 5’FL+3’AOX1 primer; Lane 3: Positive strain DNA using 5’AOX+ 3’FL primer; Lane 4: Positive strain DNA using 5’FL+3’FL;M: 100 bp ladder.

  • Southern blot of the selected transforment strain. Lane 1: Not l+EcoR I digested DNA from KM71 strain transformated from pPIC9K plasmid as the control; Lane 2: Not l+EcoR I digested pPIC9K-FL plasmid; Lane 3: Endonuclease Not I digested DNA from the selected transfórmate; Lane 4: Endonuclease EcoR I digested DNA from the selected transfórmate; Lane 5: Not l+EcoR I digested DNA from the selected transfórmate.
    Fig. 4.

    Southern blot of the selected transforment strain. Lane 1: Not l+EcoR I digested DNA from KM71 strain transformated from pPIC9K plasmid as the control; Lane 2: Not l+EcoR I digested pPIC9K-FL plasmid; Lane 3: Endonuclease Not I digested DNA from the selected transfórmate; Lane 4: Endonuclease EcoR I digested DNA from the selected transfórmate; Lane 5: Not l+EcoR I digested DNA from the selected transfórmate.

  • Northern Blot of the selected transfórmate. Lane 1: mRNA isolated from uninduced culture; Lane 2: mRNA isolated from the 24-hour inducement; Lane 3: pPIC9K/FLT-3 plasmid as the control; A: 32P labeled FLT-3 increment as the probe B: 32P labeled 18S rRNA as the probe.
    Fig. 5.

    Northern Blot of the selected transfórmate. Lane 1: mRNA isolated from uninduced culture; Lane 2: mRNA isolated from the 24-hour inducement; Lane 3: pPIC9K/FLT-3 plasmid as the control; A: 32P labeled FLT-3 increment as the probe B: 32P labeled 18S rRNA as the probe.

  • SDS-PAGE time-course of expression of FLT-3 ligand extracellular domain in the culture supernatant of recombinant P. pastoris. Samples were taken 24 h (Lane 1), 48 h (Lane 2), 72 h (Lane 3) and 96h (Lane 4) after induction with methanol. Culture supernatant (20 p.l) from each time-point was analyzed by SDS-PAGE. Lane 5 was the molecular markers.
    Fig. 6.

    SDS-PAGE time-course of expression of FLT-3 ligand extracellular domain in the culture supernatant of recombinant P. pastoris. Samples were taken 24 h (Lane 1), 48 h (Lane 2), 72 h (Lane 3) and 96h (Lane 4) after induction with methanol. Culture supernatant (20 p.l) from each time-point was analyzed by SDS-PAGE. Lane 5 was the molecular markers.

  • Western blot showing deglycosylation test of recombinant FLT-3 ligand extracellular domain. A: The protein bands on the SDS gel were visualized by Coomassi blue staining. B: recombinant proteins were partly digested with PNGase F. MW: protein molecular markers; Lane 1: PNGase F partly digested culture supernantant of induced recombinant protein; Lane 2: culture supernantant of induced recombinant protein; Lane 3: E.coli expressed recombinant FLT-3 ligand extracellular domain as the control
    Fig. 7.

    Western blot showing deglycosylation test of recombinant FLT-3 ligand extracellular domain. A: The protein bands on the SDS gel were visualized by Coomassi blue staining. B: recombinant proteins were partly digested with PNGase F. MW: protein molecular markers; Lane 1: PNGase F partly digested culture supernantant of induced recombinant protein; Lane 2: culture supernantant of induced recombinant protein; Lane 3: E.coli expressed recombinant FLT-3 ligand extracellular domain as the control

  • Recombinant FLT-3 ligand-induced proliferation of CD34* cells. The number of CD34* cells incressed 2.9 fold in the presenceof KM71pPIC9K-FL after 7 days, while in the control group it increased 1.5 fold.
    Fig. 8.

    Recombinant FLT-3 ligand-induced proliferation of CD34* cells. The number of CD34* cells incressed 2.9 fold in the presenceof KM71pPIC9K-FL after 7 days, while in the control group it increased 1.5 fold.

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