TY - JOUR
T1 - High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains : selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG
AU - Blanchard, V.
AU - Gadkari, R.A.
AU - George, A.V.E.
AU - Roy, S.
AU - Gerwig, G.J.
AU - Leeflang, B.R.
AU - Dighe, R.R.
AU - Boelens, R.
AU - Kamerling, J.P.
PY - 2008
Y1 - 2008
N2 - The methylotrophic yeast Pichia pastoris is
widely used for the production of recombinant glycoproteins.
With the aim to generate biologically active 15Nlabeled
glycohormones for conformational studies focused
on the unravelling of the NMR structures in solution, the P.
pastoris strains GS115 and X-33 were explored for the
expression of human chorionic gonadotropin (phCG) and
human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG,
produced in P. pastoris GS115, using ammonia/glycerolmethanol
as nitrogen/carbon sources, the N-glycosylation
pattern of phCG, synthesized using NH4Cl/glucose–glycerol–
methanol, comprised neutral and charged, phosphorylated
high-mannose-type N-glycans (Man8–15GlcNAc2). However,
the changed culturing protocol led to much higher amounts
of glycoprotein material, which is of importance for an
economical realistic approach of the aimed NMR research.
In the context of these studies, attention was also paid to the
site specific N-glycosylation in phCG produced in P. pastoris
GS115. In contrast to the rather simple N-glycosylation
pattern of phCG expressed in the GS115 strain, phCG and
phFSH expressed in the X-33 strain revealed, besides neutral
high-mannose-type N-glycans, also high concentrations of
neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2).
The latter finding made the X-33 strain not very suitable for
generating 15N-labeled material. Therefore, 15N-phCG was
expressed in the GS115 strain using the new optimized
protocol. The 15N-enrichment was evaluated by 15N-HSQC
NMR spectroscopy and GLC-EI/MS. Circular dichroism
studies indicated that 15N-phCG/GS115 had the same folding
as urinary hCG. Furthermore, 15N-phCG/GS115 was found
to be similar to the unlabeled protein in every respect as
judged by radioimmunoassay, radioreceptor assays, and in
vitro bioassays.
Keywords Pichia pastoris
AB - The methylotrophic yeast Pichia pastoris is
widely used for the production of recombinant glycoproteins.
With the aim to generate biologically active 15Nlabeled
glycohormones for conformational studies focused
on the unravelling of the NMR structures in solution, the P.
pastoris strains GS115 and X-33 were explored for the
expression of human chorionic gonadotropin (phCG) and
human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG,
produced in P. pastoris GS115, using ammonia/glycerolmethanol
as nitrogen/carbon sources, the N-glycosylation
pattern of phCG, synthesized using NH4Cl/glucose–glycerol–
methanol, comprised neutral and charged, phosphorylated
high-mannose-type N-glycans (Man8–15GlcNAc2). However,
the changed culturing protocol led to much higher amounts
of glycoprotein material, which is of importance for an
economical realistic approach of the aimed NMR research.
In the context of these studies, attention was also paid to the
site specific N-glycosylation in phCG produced in P. pastoris
GS115. In contrast to the rather simple N-glycosylation
pattern of phCG expressed in the GS115 strain, phCG and
phFSH expressed in the X-33 strain revealed, besides neutral
high-mannose-type N-glycans, also high concentrations of
neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2).
The latter finding made the X-33 strain not very suitable for
generating 15N-labeled material. Therefore, 15N-phCG was
expressed in the GS115 strain using the new optimized
protocol. The 15N-enrichment was evaluated by 15N-HSQC
NMR spectroscopy and GLC-EI/MS. Circular dichroism
studies indicated that 15N-phCG/GS115 had the same folding
as urinary hCG. Furthermore, 15N-phCG/GS115 was found
to be similar to the unlabeled protein in every respect as
judged by radioimmunoassay, radioreceptor assays, and in
vitro bioassays.
Keywords Pichia pastoris
M3 - Article
SN - 0282-0080
VL - 25
SP - 245
EP - 257
JO - Glycoconjugate Journal
JF - Glycoconjugate Journal
ER -