Typically one would immunoprecipitate or try to column purify the antigen and try to get a partial sequence first. Is there now a "library screening method" available for the quick identification of the target of a new antibody?
maybe you can find answers here
Biotechnol. Prog. 2008, 24, 561-569
mRNA-Display-Based Selections for Proteins with Desired Functions: A
Protease-Substrate Case Study
C. Alexander Valencia, Steven W. Cotten, Biao Dong, and Rihe Liu*
School of Pharmacy and Carolina Center for Genomic Sciences, The University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599
mRNA-display is an amplification-based, iterative rounds of in Vitro protein selection techniquethat circumvents a number of difficulties associated with yeast two-hybrid and phage display.Because of the covalent linkage between the genotype and the phenotype, mRNA-display providesa powerful means for reading and amplifying a peptide or protein sequence after it has beenselected from a library with a diversity in the range of 1012-1013. In this paper, we brieflyreview the recent progress in using mRNA-display to identify affinity reagents, binding partners,and enzyme substrates from synthetic peptide or natural proteome libraries. To facilitate the use
of mRNA-display in research laboratories without previous experience, we provide a detailedanalysis of the critical steps of an mRNA-display-based selection in a case study for theidentification of the natural substrates of caspases, including the generation of an mRNA-displayedproteome library, removal of abundant sequences, and selection of proteins with desired functions.The advantages and technical limitations of mRNA-display as a general peptide or proteinselection tool are also addressed.IntroductionmRNA-display is an in Vitro selection technique that allows
for the identification of polypeptide sequences with desiredproperties from either a natural protein library or a combinatorial peptide library (1-3). The central feature of this method is that the polypeptide chain is covalently linked to the 3¢ end of its
own mRNA. This is accomplished by synthesis and in Vitro translation of an mRNA template with puromycin attached to its 3¢ end via a short DNA linker. During in Vitro translation, when the ribosome reaches the RNA-DNA junction and translation pauses, puromycin, an antibiotic that mimics the aminoacyl moiety of tRNA, enters the ribosome “A” site and accepts the nascent polypeptide by forming a peptide bond. This results in tethering the nascent peptide to its own mRNA. Since the genotype coding sequence and the phenotype polypeptide sequence are united in a single molecule, mRNA-display provides a powerful means for reading and amplifying a peptide or protein sequence after it has been selected on the basis of its
function. Multiple rounds of selection and amplification can be performed, enabling enrichment of rare sequences with desiredproperties. Compared to prior peptide or protein selection
methods, mRNA-display has several major advantages. First,
the genotype is covalently linked to and is always present with
the phenotype. This stable linkage makes it possible to use any
arbitrary conditions in the functional selection with tunable
stringency. Second, the complexity of the peptide or protein
library can be close to that of the RNA or DNA pools. Peptide
or protein libraries containing as many as 1012-1013 unique
sequences can be readily generated and selected, a few orders
of magnitude higher than can be achieved using phage display.
Therefore, both the likelihood of isolating rare sequences and
the diversity of the sequences isolated in a given selection are
significantly increased.
In principle, mRNA-display can be used in any selection that
aims at the identification of peptide or protein sequences with
desired functions. So far, mRNA-display has been successfully
applied in the improvement of the binding affinities of existing
affinity reagents, identification of drug-binding targets, mapping
of the protein-protein interaction and DNA-protein interaction
networks, and most recently, elucidation of the proteasesubstrate
interactions (4-11). The various applications of
mRNA-display can be grouped based on the type library and
the selection parameters. Figure 1 illustrates a general selection
scheme for the identification of the binding partners of a target
of interest, whereas Figure 2 shows the general procedure for
the selection of the downstream substrates of a protease of
interest. Here we briefly review the current progress of using
mRNA-display in the selection of peptide or proteins sequences
with desired functions, including those from synthetic peptide
and natural proteome libraries.
Selection of Polypeptides with Desired Functions from
Synthetic Polypeptide Libraries
The first type of library that can be used for mRNA-displaybased
selection is a synthetic combinatorial library that contains
varying length of totally or partially randomized amino acids.
Using a 108-residue synthetic library containing random peptides
patterned to form R-helices, â-strands, and unpatterened random
sequences, Wilson and co-workers were able to isolate more
than 20 peptides that bound to streptavidin with dissociation
constants as low as 5 nM after seven rounds of selection from
a pool of 6.7 1012 unique sequences (12). Similarly, Keefe
and Szostak used a 108-residue synthetic library containing 80
totally randomized amino acids to identify polypeptides that
bound to small molecule ATP. After eight rounds of selection
from a pool of 6 1012 unique sequences, four unrelated classes
of ATP-binding polypeptides were isolated (13). The binding
of these sequences to ATP is specific, and the optimized
but nowadays, if you are working in models systems (human others), think about getting a few peptide by mass spec, and after there is a good chance you will have access to the cDNA throught resources (ORFeome in human, mouse etc)
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