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Genotyping of recombinant Pichia pastoris strains

Abstract

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.

Abbreviations

AFLP:

amplified-fragment length polymorphism

AOX1:

alcohol oxidase 1 gene

BMG:

growth medium: 100 mM potassium phosphate, pH 6.0, 0.4 mg/l biotin, 1.34% YNB, 1% glycerol

BMM:

expression medium: 100 mM potassium phosphate, pH 6.0, 0.4 mg/l biotin, 1.34% YNB, 0.75% (v/v) methanol

DMT-off:

without dimethoxytrityl

n.d.:

not determined

PCR:

polymerase chain reaction

RDB:

1 M sorbitol, 1.34% YNB, 2% dextrose, 0.4 mg/l biotin, 0.005% amino acids without histidine

r2 :

coefficient of determination

RP:

recombinant protein

SDS:

sodium dodecyl sulphate

TE buffer:

10 mM Tris-Cl, 1 mM EDTA, pH 8.0

TP:

total protein

XA-1 and XA-2:

XhoI site cohesive-ended complementary oligonucleotides to generate the XhoI restriction fragment adapter

XP-G:

PCR primer used in the AFLP reactions directed at the adapter tail and with a G as selective nucleotide at the 3′-end

YNB:

yeast nitrogen base

YPD:

1% yeast extract, 2% peptone, 2% dextrose

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Correspondence to José M. Viader-Salvadó.

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Viader-Salvadó, J.M., Cab-Barrera, E.L., Galán-Wong, L.J. et al. Genotyping of recombinant Pichia pastoris strains. Cell Mol Biol Lett 11, 348–359 (2006). https://doi.org/10.2478/s11658-006-0029-z

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Key words

  • Pichia pastoris
  • Amplified-fragment length polymorphism (AFLP)
  • Clonal variation