In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries.
The covalent coupling of an mRNA to the protein that it encodes (mRNA display) provides a powerful tool for analysis of protein function in the post-genomic era. This coupling allows the selective enrichment of individual members from libraries of displayed proteins and the subsequent regeneration of an enriched library using the RNA moiety. Tissue-specific libraries from poly(A)(+) mRNA were prepared by priming first and second strand cDNA synthesis with oligonucleotides containing nine random 3' nucleotides, the fixed regions of which encoded the requisite sequences for formation of mRNA display constructs and a library-specific sequence tag. Starting with a pool of uniquely tagged libraries from different tissues, an iterative selection was performed for binding partners of the anti-apoptotic protein Bcl-X(L). After four rounds of selection, the pool was deconvoluted by polymerase chain reaction amplification with library-specific primers. Subsequent clonal sequence analysis revealed the selection of three members of the Bcl-2 family known to bind to Bcl-X(L). In addition, several proteins not previously demonstrated to interact with Bcl-X(L) were identified. The relative binding affinities of individual selected peptides were determined, as was their susceptibility to competition with a BH3 domain peptide. Based on these data, a putative BH3 domain was identified in most peptides.
Phylos, Inc., Lexington, Massachusetts 02421, USA. phammond@phylos.com
P W
PW
Hammond
J
J
Alpin
C E
CE
Rise
M
M
Wright
B L
BL
Kreider
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D019992:Databases as Topic; D020224:Expressed Sequence Tags; D015723:Gene Library; D016259:Genes, myc; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein