Generation and characterization of a chimeric rabbit/human Fab for co-crystallization of HIV-1 Rev.
Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation, which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single-chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against human immunodeficiency virus type 1.
Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-2775, USA.
Stephen J
SJ
Stahl
Norman R
NR
Watts
Christoph
C
Rader
Michael A
MA
DiMattia
Rose G
RG
Mage
Ira
I
Palmer
Joshua D
JD
Kaufman
Jonathan M
JM
Grimes
David I
DI
Stuart
Alasdair C
AC
Steven
Paul T
PT
Wingfield
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D012367:RNA, Viral / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology*
D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus