Re: Aptein & polysome display (patents & technology)
Press release regarding Aptein patents
Aptein Inc., has been issued two United States patents, entitled "Cell-Free Synthesis" and "Isolation of Novel Genes and Polypeptides," that broadly cover methods to screen over 10^12 random polypeptides in small volumes. These patents describe a technology that utilizes in vitro translation to produce large libraries of "polysomes" that allow for the recovery of high-affinity binders against receptors and other targets. This cell-free process involves polysome particles that contain a randomly generated polypeptide plus the genetic material coding for the peptide (mRNA), and one or more ribosomes engaged in protein synthesis. The polysomes bind via the peptides or proteins to targets of pharmaceutical interest and the associated genes can be rescued by gene amplification. A single polypeptide binding event may be detected and the sequence may be recovered with this invention. Typically, three or four rounds of polysome selection are serially performed to enrich for the highest affinity molecules, which are identified by DNA sequencing and binding assays. The terms, "polysome display" and "ribosome display," have been used to describe the same process.
Some info for self education fom Medline:
1
NLM CIT. ID: 94377484
TITLE: An in vitro polysome display system for identifying ligands from very
large peptide libraries.
AUTHORS: Mattheakis LC; Bhatt RR; Dower WJ
AUTHOR AFFILIATION:
Affymax Research Institute, Palo Alto, CA 94304.
PUBLICATION TYPES:
JOURNAL ARTICLE
LANGUAGES:
Eng
REGISTRY NUMBERS:
0 (Antibodies, Monoclonal)
0 (DNA Primers)
0 (Endorphins)
0 (Epitopes)
0 (Ligands)
0 (Peptides)
0 (Recombinant Fusion Proteins)
74913-18-1 (Dynorphins)
83335-41-5 (rimorphin)
ABSTRACT:
We have used an in vitro protein synthesis system to construct a very
large library of peptides displayed on polysomes. A pool of DNA
sequences encoding 10(12) random decapeptides was incubated in an
Escherichia coli S30 coupled transcription/translation system.
Polysomes were isolated and screened by affinity selection of the
nascent peptides on an immobilized monoclonal antibody specific for
the peptide dynorphin B. The mRNA from the enriched pool of polysomes
was recovered, copied into cDNA, and amplified by the polymerase
chain reaction (PCR) to produce template for the next round of in
vitro synthesis and selection. A portion of the amplified template
from each round was cloned into a filamentous phagemid vector to
determine the specificity of peptide binding by phage ELISA and to
sequence the DNA. After four rounds of affinity selection, the
majority of clones encoded peptides that bound specifically to the
antibody and contained a consensus sequence that is similar to the
known epitope for the antibody. Synthetic peptides corresponding to
several of these sequences have binding affinities ranging from 7 to
140 nM. The in vitro system described here has the potential to
screen peptide libraries that are three to six orders of magnitude
larger than current biological peptide display systems.
NLM PUBMED CIT. ID:
7522328
SOURCE: Proc Natl Acad Sci U S A 1994 Sep 13;91(19):9022-6 |
