Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression
For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applic...
Ausführliche Beschreibung
Autor*in: |
Meng Mei [verfasserIn] Junhong Li [verfasserIn] Shengchen Wang [verfasserIn] Ki Baek Lee [verfasserIn] Brent L. Iverson [verfasserIn] Guimin Zhang [verfasserIn] Xin Ge [verfasserIn] Li Yi [verfasserIn] |
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Format: |
E-Artikel |
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Sprache: |
Englisch |
Erschienen: |
2019 |
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Übergeordnetes Werk: |
In: Frontiers in Bioengineering and Biotechnology - Frontiers Media S.A., 2014, 7(2019) |
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Übergeordnetes Werk: |
volume:7 ; year:2019 |
Links: |
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DOI / URN: |
10.3389/fbioe.2019.00362 |
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Katalog-ID: |
DOAJ044601972 |
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10.3389/fbioe.2019.00362 doi (DE-627)DOAJ044601972 (DE-599)DOAJ6175a1fe8a844265bb39d6a010c182ba DE-627 ger DE-627 rakwb eng TP248.13-248.65 Meng Mei verfasserin aut Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries. yeast surface display Fab divergent promotor ER retention sequence molecular chaperone Biotechnology Junhong Li verfasserin aut Shengchen Wang verfasserin aut Ki Baek Lee verfasserin aut Brent L. Iverson verfasserin aut Guimin Zhang verfasserin aut Xin Ge verfasserin aut Li Yi verfasserin aut In Frontiers in Bioengineering and Biotechnology Frontiers Media S.A., 2014 7(2019) (DE-627)74950403X (DE-600)2719493-0 22964185 nnns volume:7 year:2019 https://doi.org/10.3389/fbioe.2019.00362 kostenfrei https://doaj.org/article/6175a1fe8a844265bb39d6a010c182ba kostenfrei https://www.frontiersin.org/article/10.3389/fbioe.2019.00362/full kostenfrei https://doaj.org/toc/2296-4185 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2019 |
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10.3389/fbioe.2019.00362 doi (DE-627)DOAJ044601972 (DE-599)DOAJ6175a1fe8a844265bb39d6a010c182ba DE-627 ger DE-627 rakwb eng TP248.13-248.65 Meng Mei verfasserin aut Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression 2019 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries. yeast surface display Fab divergent promotor ER retention sequence molecular chaperone Biotechnology Junhong Li verfasserin aut Shengchen Wang verfasserin aut Ki Baek Lee verfasserin aut Brent L. Iverson verfasserin aut Guimin Zhang verfasserin aut Xin Ge verfasserin aut Li Yi verfasserin aut In Frontiers in Bioengineering and Biotechnology Frontiers Media S.A., 2014 7(2019) (DE-627)74950403X (DE-600)2719493-0 22964185 nnns volume:7 year:2019 https://doi.org/10.3389/fbioe.2019.00362 kostenfrei https://doaj.org/article/6175a1fe8a844265bb39d6a010c182ba kostenfrei https://www.frontiersin.org/article/10.3389/fbioe.2019.00362/full kostenfrei https://doaj.org/toc/2296-4185 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2019 |
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Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression |
abstract |
For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries. |
abstractGer |
For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries. |
abstract_unstemmed |
For antibody discovery and engineering, yeast surface display (YSD) of antigen-binding fragments (Fabs) and coupled fluorescence activated cell sorting (FACS) provide intact paratopic conformations and quantitative analysis at the monoclonal level, and thus holding great promises for numerous applications. Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. The feasibility of affinity maturation was further demonstrated by isolating a high affinity Fab clone from 1:103 or 1:105 spiked libraries. |
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title_short |
Prompting Fab Yeast Surface Display Efficiency by ER Retention and Molecular Chaperon Co-expression |
url |
https://doi.org/10.3389/fbioe.2019.00362 https://doaj.org/article/6175a1fe8a844265bb39d6a010c182ba https://www.frontiersin.org/article/10.3389/fbioe.2019.00362/full https://doaj.org/toc/2296-4185 |
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Using anti-TNFα mAbs Infliximab, Adalimumab, and its variants as model Fabs, this study systematically characterized complementary approaches for the optimization of Fab YSD. Results suggested that by using divergent promoter GAL1-GAL10 and endoplasmic reticulum (ER) signal peptides for co-expression of light chain and heavy chain-Aga2 fusion, assembled Fabs were functionally displayed on yeast cell surface with sigmoidal binding responses toward TNFα. Co-expression of a Hsp70 family molecular chaperone Kar2p and/or protein-disulfide isomerase (Pdi1p) significantly improved efficiency of functional display (defined as the ratio of cells displaying functional Fab over cells displaying assembled Fab). Moreover, fusing ER retention sequences (ERSs) with light chain also enhanced Fab display quality at the expense of display quantity, and the degree of improvements was correlated with the strength of ERSs and was more significant for Infliximab than Adalimumab. 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