Abstract
The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275-1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage λ libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 111-117 |
| Number of pages | 7 |
| Journal | Gene |
| Volume | 128 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jun 15 1993 |
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Keywords
- affinity-tail sequence
- Artificial operon
- coliphage T3 promoter
- combinatorial library
- monocistronic expression
- plaque-lift assay
ASJC Scopus subject areas
- Genetics
Cite this
A revised strategy for cloning antibody gene fragments in bacteria. / Posner, B.; Lee, I.; Itoh, T.; Pyati, J.; Graff, R.; Thorton, G. B.; La Polla b, R.; Benkovic, S. J.
In: Gene, Vol. 128, No. 1, 15.06.1993, p. 111-117.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A revised strategy for cloning antibody gene fragments in bacteria
AU - Posner, B.
AU - Lee, I.
AU - Itoh, T.
AU - Pyati, J.
AU - Graff, R.
AU - Thorton, G. B.
AU - La Polla b, R.
AU - Benkovic, S. J.
PY - 1993/6/15
Y1 - 1993/6/15
N2 - The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275-1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage λ libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.
AB - The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275-1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage λ libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.
KW - affinity-tail sequence
KW - Artificial operon
KW - coliphage T3 promoter
KW - combinatorial library
KW - monocistronic expression
KW - plaque-lift assay
UR - http://www.scopus.com/inward/record.url?scp=0027245979&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027245979&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(93)90161-U
DO - 10.1016/0378-1119(93)90161-U
M3 - Article
C2 - 8508951
AN - SCOPUS:0027245979
VL - 128
SP - 111
EP - 117
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1
ER -