Abstract
Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193-9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.
| Original language | English |
|---|---|
| Pages (from-to) | 1385-1391 |
| Number of pages | 7 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 88 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 2010 Dec 1 |
| Externally published | Yes |
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Keywords
- Anchored periplasmic expression
- Antigens on the human cell surface
- ScFv
- Spheroplasts
ASJC Scopus subject areas
- Biotechnology
- Applied Microbiology and Biotechnology
Cite this
Enrichment of Escherichia coli spheroplasts displaying scFv antibodies specific for antigens expressed on the human cell surface. / Qiu, Jun Kang; Jung, Sang Taek; Georgiou, George; Hang, Hai Ying.
In: Applied Microbiology and Biotechnology, Vol. 88, No. 6, 01.12.2010, p. 1385-1391.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Enrichment of Escherichia coli spheroplasts displaying scFv antibodies specific for antigens expressed on the human cell surface
AU - Qiu, Jun Kang
AU - Jung, Sang Taek
AU - Georgiou, George
AU - Hang, Hai Ying
PY - 2010/12/1
Y1 - 2010/12/1
N2 - Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193-9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.
AB - Anchored periplasmic expression (APEx) is a method for isolating high affinity ligand-binding proteins from large combinatorial libraries, and antibodies highly specific for soluble antigens were successfully isolated from APEx antibody libraries in combination with flow cytometric sorting (Harvey et al., Proc Natl Acad Sci USA 101(25):9193-9198, 2004). However, many disease markers and drug targets are localized on the cell surface, and often, unique posttranslational modifications and/or properly folded epitopes are lost when they were expressed and isolated in soluble form. In this study, we demonstrate that Escherichia coli spheroplasts, displaying antibodies and screened by a combination of plate-panning and flow cytometric sorting, can be used for isolating antibodies specific for antigens on the human cell surface. Two rounds of plate-panning followed by one round of flow cytometric sorting resulted in 7,200-fold enrichment of antibodies specific for the protective antigen of Bacillus anthracis from a large excess of spheroplasts expressing a scFv antibody to digoxin (a negative control). There is the potential to use this technique for library screening to find novel antibodies against disease cell surface antigens.
KW - Anchored periplasmic expression
KW - Antigens on the human cell surface
KW - ScFv
KW - Spheroplasts
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U2 - 10.1007/s00253-010-2861-3
DO - 10.1007/s00253-010-2861-3
M3 - Article
VL - 88
SP - 1385
EP - 1391
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 6
ER -