Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris

1999 ◽  
Vol 52 (3) ◽  
pp. 410-414 ◽  
Author(s):  
Y. Sakuma ◽  
M. Kimura ◽  
T. Takabatake ◽  
K. Takeshima ◽  
H. Fujimura
Keyword(s):  
Pichia Pastoris ◽  
Sonic Hedgehog ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris

2003 ◽  
Vol 30 (2) ◽  
pp. 213-225 ◽  
Author(s):  
AE Fidler ◽  
JS Lin ◽  
S Lun ◽  
W Ng Chie ◽  
A Western ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

Expression of Biologically Active Recombinant Pokeweed Antiviral Protein in Methylotrophic Yeast Pichia pastoris

2000 ◽  
Vol 18 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Francis Rajamohan ◽  
Sekou O. Doumbia ◽  
Cherri R. Engstrom ◽  
Sharon L. Pendergras ◽  
Daniell L. Maher ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Pokeweed Antiviral Protein ◽  
Antiviral Protein ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris

2002 ◽  
Vol 14 (6) ◽  
pp. 327 ◽  
Author(s):  
Andrew E. Fidler ◽  
Andrea H. Western ◽  
Nicole Griffith ◽  
Lynne Selwood ◽  
Vicki Stent ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Germ Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Brushtail Possum ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians. To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris. The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCF. The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro. Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF. This source of biologically active marsupial SCF may prove useful in future studies of marsupial development.

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.

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