In Vitro Selection and Characterization of Bcl-XL-binding Proteins from a Mix of Tissue-specific mRNA Display Libraries

Philip W Hammond,Julia Alpin,Cecil E Rise,Martin Wright,Brent L Kreider

doi:10.1074/jbc.m011641200

Abstract

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.

Highlights

The Bcl-2 family proteins containing multiple BH domains may function in part through the regulation of mitochondrial membrane potential and a corresponding release of cytochrome c into the cytoplasm
Choice of UTR Sequence Tags—In order to increase the diversity of the pool of mRNA displayed proteins used in the selection, it was desirable to mix together libraries prepared from different tissue sources
Model Binding Study—In order to demonstrate the feasibility of using mRNA display technology to identify proteins that bind to Bcl-XL, a model study was performed using known BH3 domains

Summary

EXPERIMENTAL PROCEDURES

Choice of UTR Sequence Tags—In order to increase the diversity of the pool of mRNA displayed proteins used in the selection, it was desirable to mix together libraries prepared from different tissue sources. The c-myc construct described by Roberts and Szostak [13] was amplified by PCR using the 5Ј primer TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT HHH HHH HHA CAA TGG CTG AAG AAC AGA AAC TG (where H is an equimolar mixture of A, C, and T) This inserted 8 random bases into the 5Ј-UTR upstream of the ATG start codon, to give a library of 38 (6561) different mRNA molecules after in vitro transcription with T7 RNA polymerase. 60 ␮g of Bcl-XL-GST fusion protein (100 ␮l) was added, allowed to bind for 1 h at 4 °C, and the beads rewashed in selection buffer (50 mM Tris-HCl (pH 7.5), 150 mM KCl, 0.05% Triton X-100, 0.5 mg/ml bovine serum albumin, 0.1 mg/ml salmon sperm DNA). After translation in rabbit reticulocyte lysate (Ambion) peptides were purified directly from the lysate by immunoprecipitation and peptide elution based on a C-terminal FLAG-M2 epitope (Sigma)

RESULTS

Novel protein D

Accession number

Total clones

DISCUSSION