Abstract
High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His6 epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.
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Abbreviations
- scFv:
-
single-chain antibody
- VH:
-
immunoglobulin heavy chain variable region
- VL:
-
immunoglobulin light chain variable region
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- ELISA:
-
enzyme-linked immunosorbent assay
- IMAC:
-
immobilized metal ion chromatography
- ORF:
-
open reading frame
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Shea, C., Bloedorn, L. & Sullivan, M.A. Rapid Isolation of Single-chain Antibodies for Structural Genomics. J Struct Funct Genomics 6, 171–175 (2005). https://doi.org/10.1007/s10969-005-5246-6
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DOI: https://doi.org/10.1007/s10969-005-5246-6