Selection of Pichia pastoris strains expressing recombinant immunoglobulin G by cell surface labeling

A simple cell labeling method for sorting yeast Pichia pastoris antibody expressing strains is described. A small portion of secreted recombinant antibody retained on the cell surface was labeled with fluorescence detection antibody. The signal intensity of the labeled cell was correlated with the c...

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Bibliographic Details
Published in:Journal of immunological methods 2010-06, Vol.358 (1), p.66-74
Main Authors: Lin, Song, Shen, Zheng, Zha, Dongxing, Sharkey, Nathan, Prinz, Bianka, Hamilton, Stephen, Pavoor, Tej Venkatachalam, Bobrowicz, Beata, Shaikh, Seemab S., Rittenhour, Alissa M., Potgieter, Thomas I., Bobrowicz, Piotr, Stadheim, Terrance A.
Format: Article
Language:English
Subjects:
Animals
Antibodies - immunology
Bioreactors
Cell surface labeling
Flow Cytometry
Fluorescence activated cell sorting (FACS)
Goats
Humans
Immunoglobulin G - biosynthesis
Immunoglobulin G - genetics
Immunoglobulin G - immunology
Membrane Proteins - analysis
Membrane Proteins - genetics
Membrane Proteins - immunology
Membrane Proteins - metabolism
Microscopy, Confocal
Pichia - classification
Pichia - cytology
Pichia - isolation & purification
Pichia - metabolism
Pichia pastoris
Pichia pastoris antibody expressing strain
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - immunology
Recombinant Proteins - analysis
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Staining and Labeling - methods
Strain productivity improvement
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Description
Summary:A simple cell labeling method for sorting yeast Pichia pastoris antibody expressing strains is described. A small portion of secreted recombinant antibody retained on the cell surface was labeled with fluorescence detection antibody. The signal intensity of the labeled cell was correlated with the cell's antibody productivity. Using this labeling technique to sort a mixture model induced in the same fermenter where the cells of high producing strain were spiked into a population of a low producing strain at the frequency of 1:100,000, one round of sorting achieved a ∼ 5000-fold enrichment of the high producing strain. A variety of P. pastoris strains expressing antibody sorted based on the signal intensity on the cell surface yielded titer improvements by 30% to 300%. Our data demonstrate that Pichia cell surface labeling is a simple, effective and reliable method for sorting Pichia antibody expressing strains for productivity improvement.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2010.03.007