Journal: Microbial Cell Factories

Article Title: Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

doi: 10.1186/1475-2859-10-37

Figure Lengend Snippet: Expression of His-tagged MeH in P. pastoris transformants induced with methanol . (A) SDS-PAGE of whole cell lysates from P. pastoris transformants carrying empty vector or multicopy MeH expression cassetes. (B) Western blotting of the same samples using anti-tetraHis antibody. M- prestained protein marker, C- Control (pPIC3.5K Mut S ), 1,2,3- multicopy pPIC3.5K-MeH transformants for intracellular expression of MeH, resistant to various concentrations of antibiotic G418. Solid arrows indicate unglycosylated MeH polypeptide precursor, dotted arrows point to glycosylated MeH form and dashed arrows show cellular proteins, upregulated in response to MeH expression.

Article Snippet: Both MuHN and MeH genes were also cloned into the Avr II site of P. pastoris vector pPIC3.5K (Invitrogen) for intracellular expression under the control of methanol inducible AOX1 promoter.

Techniques: Expressing, SDS Page, Plasmid Preparation, Western Blot, Marker

Journal: Microbial Cell Factories

Article Title: Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

doi: 10.1186/1475-2859-10-37

Figure Lengend Snippet: Analysis of MeH in insoluble protein fractions from P. pastoris by Western blotting using anti-His antibody . The fractions insoluble in 1% TritonX-100 and 1M NaCl were obtained from P. pastoris transformants using the same method as described in Methods for S. cerevisiae samples (correspond to fraction 7 in Figure 3). VK indicates the sample obtained from P. pastoris transformant carrying one copy of pPIC3.5K-MeH expression cassete. Other markings are the same as in Figure 5 (dotted arrow indicates glycosylated MeH form, whereas solid arrow - unglycosylated MeH precursor).

Article Snippet: Both MuHN and MeH genes were also cloned into the Avr II site of P. pastoris vector pPIC3.5K (Invitrogen) for intracellular expression under the control of methanol inducible AOX1 promoter.

Techniques: Western Blot, Expressing

Journal: Microbial Cell Factories

Article Title: Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

doi: 10.1186/1475-2859-10-37

Figure Lengend Snippet: Analysis of MeH expression in whole cell lysates from P. pastoris and S. cerevisiae . Whole cell lysates from P. pastoris (C, In, SP and SG) and S. cerevisiae (Sc) were analysed by Western blotting using anti-His antibody. M- marker, C- control, In- multicopy pPIC3.5K-MeH transformant for intracellular expression of full sequence MeH protein, SP- multicopy pPIC9K-MeH transformant for secreted expression of chimeric α-MeH protein with α-factor signal sequence (overexpressing both polypeptide precursor and glycosylated MeH forms), SG- another multicopy pPIC9K-MeH transformant for secreted expression of chimeric α-MeH, overexpressing glycosylated MeH form, Sc- pFGG3-MeH transformant of S. cerevisiae AH22 strain overexpressing full sequence MeH protein after induction with galactose. Solid arrows indicate unglycosylated MeH polypeptides, whereas dotted arrows - glycosylated MeH forms.

Article Snippet: Both MuHN and MeH genes were also cloned into the Avr II site of P. pastoris vector pPIC3.5K (Invitrogen) for intracellular expression under the control of methanol inducible AOX1 promoter.

Techniques: Expressing, Western Blot, Marker, Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-Throughput Quantitation of Yeast uORF Regulatory Impacts Using FACS-uORF

doi: 10.1007/978-1-0716-1851-6_18

Figure Lengend Snippet:

Article Snippet: 19 FastDigest XmaJI (Cuts AvrII sites (isoschizomer)) (ThermoFisher Scientific).

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-Throughput Quantitation of Yeast uORF Regulatory Impacts Using FACS-uORF

doi: 10.1007/978-1-0716-1851-6_18

Figure Lengend Snippet:

Article Snippet: 19 FastDigest XmaJI (Cuts AvrII sites (isoschizomer)) (ThermoFisher Scientific).

Techniques: Ligation