Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris

2001 ◽  
Vol 442 (7) ◽  
pp. r184-r186 ◽  
Author(s):  
Marija Anžur Lasnik ◽  
Vladka Gaberc Porekar ◽  
Anton štalc
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Stimulating Factor

scholarly journals Cloning and expression of human granulocyte-macrophage colony stimulating factor (hGM-CSF) in Pichia pastoris

2013 ◽  
Vol 16 (3) ◽  
pp. 22-29
Author(s):  
Lien Thi Kim Phan ◽  
Truong Tat Dang ◽  
Hieu Van Tran
Keyword(s):  
Pichia Pastoris ◽  
Shuttle Vector ◽  
Methylotrophic Yeast ◽  
Cloning And Expression ◽  
Colony Stimulating Factor ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Cloned Gene ◽  
Stimulating Factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein, belongs to the family of colony stimulating factors (CSFs) that regulate proliferation and differentiation of hematopoietic cells. Recombinant hGM-CSF (rhGM-CSF) is one of FDA-appoved, therapeutic recombinant proteins that have been successfully used to treat many diseases like neutropenia, leukemia, or in combination with chemical therapy, etc. In this study, we reported the production of rhGM-CSF in methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. The gene hgm-csf encoding for hGM-CSF comprising of 415 bps was isolated using PCR reaction with two primers hGM-F and hGM-R bearing XhoI and NotI restriction sites, respectively. After double digesting with these enzymes, the hgm-csf fragment was cloned into pPICZαA shuttle vector, between the XhoI and NotI sites. Recombinant vector containing the gene hgm-csf, termed pPICZαA/hGMCSF, under the control of promoter AOX has the ability to express hGM-CSF in P. pastoris. The accuracy of cloning strategy was confirmed by digestion with REs, PCR and sequencing. Sequencing alignment showed that the cloned gene completely homologous to those published on Genebank. SacI linearized pPICZαA/hGM-CSF was transformed into P. pastoris X33 strain to establish the recombinant P. pastoris X33::hgm-csf. In methanol-contained, inducing BMMY medium, the recombinant X33 cells expressed and secreted hGM-CSF into the supernatant. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a nonglycosylated form on SDS-PAGE gel. This result was confirmed by Western blot with specific antibodies.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

scholarly journals Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

2013 ◽  
Vol 1831 (2) ◽  
pp. 282-290 ◽  
Author(s):  
Vasyl A. Ivashov ◽  
Karlheinz Grillitsch ◽  
Harald Koefeler ◽  
Erich Leitner ◽  
Dominic Baeumlisberger ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Lipid Droplets ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia pastoris using Agilent 2100 Bioanalyzer

2009 ◽  
Vol 143 (1) ◽  
pp. 44-50 ◽  
Author(s):  
Anjali Apte-Deshpande ◽  
Sandeep Somani ◽  
Goutam Mandal ◽  
Sudheerbabu Soorapaneni ◽  
Sriram Padmanabhan
Keyword(s):  
Pichia Pastoris ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Over Expression ◽  
Stimulating Factor
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