Rapid isolation of single-chain antibodies for structural genomics.
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 His(6) epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.
Shea
Colleen
C
Department of Pediatrics and Center for Human Genetics and Molecular Pediatric Disease, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, NY 14642, USA.
Bloedorn
Laura
L
Sullivan
Mark A
MA
D000818:Animals; D002852:Chromatography, Ion Exchange; D003460:Crystallization / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification* / Q000378:metabolism; D018320:Leishmania major / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015800:Protozoan Proteins / Q000302:isolation & purification* / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D046912:Multiprotein Complexes; D015800:Protozoan Proteins