Isolating biological modulators from biodiverse gene fragment libraries

Abstract

The present invention provides a method for identifying a modulator or mediator of a biological activity, which activity includes antigenicity and or immunogenicity, said method comprising the step of: (i) producing a gene fragment expression library derived from defined nucleotide sequence fragments; and (ii) assaying the expression library for at least an amino acid sequence derived from step (i) for a biological activity wherein that activity is different from any activity the amino acid sequence may have in its native environment.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the field of screening gene libraries, and more particularly to the generation and screening of natural domain libraries derived from organisms with known genomic sequences. Methods for increasing the diversity of such biodiverse gene fragment libraries further by mutagenesis procedures are described. The present invention also provides the means by which a wide range of peptide-based therapeutics, prophylactics and diagnostic reagents may be developed. General [0002] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variation and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations or any two or mor...

AI Technical Summary

Benefits of technology

[0035] Proteins of different function show evidence of evolving by shuffling of domains (eg. nerve growth factor and the low-density lipoprotein receptors) or by minor modifications of different residues within conserved domains (serine proteases). The present invention seeks to mimic this evolution by using peptide libraries encoded by known and defined nucleotide sequence fragments that are a rich source of peptides containing amino acid sequences evolved for diverse molecular interactions not necessarily closely related to the function performed within the donor organism. Also described are means of extending the diversity of biodiverse gene fragment libraries further by mutagenesis—either in vitro using PCR amplification under mutagenic conditions, or in vivo by replication of the library in ‘mutator’ bacterial strains which contain mutations in genes involved in mismatch repair of DNA.

[0041] A distinct advantage of the technology described herein is that through having greater control over the composition of an amino acid sequence expression library by knowing its defined constitution, one can intentionally maximise the phylogenetic distance between the constituent genomes of the library to ensure a maximal degree of diversity which, could in principle rival the sequence diversity of environmentally derived genome samples, notwithstanding the fact that such samples may contain more species diversity per se. This approach will become increasingly powerful as the range of available nucleotide sequences increase further.

Problems solved by technology

In other instances the activity can be dependent on the conformation of complete polypeptide and cannot be obtained by these techniques.

The ability to isolate active peptides from random fragment libraries can however be highly variable and problems with low affinity interactions have been reported, particularly for peptides required to represent complex conformations such as discontinuous epitopes bound by many antibodies.

There is unpredictability in that, libraries that are a rich source of peptides for one ligand may not contain peptides for others.

While the ability to obtain desired peptides should be increased with libraries containing larger random peptides and more random peptides there are practical difficulties in conducting high throughput screening or affinity purification particularly since it has been shown that high-density affinity purification is inefficient.

There is also uncertainty about the degree to which peptides isolated from the random peptide libraries will retain their binding or biological activity when produced as part of different delivery strategies such as fusions with different polypeptides.

However, a major problem to be overcome in the field of peptide therapeutics and prophylactics is the identification of specific amino acid sequences having a desired antagonist or agonist activity against a particular biological activity in a particular cellular environment.

Such candidate peptide drugs may be particularly difficult to identify from truly random peptide libraries that lack any enrichment for sequences encoding molecular shapes suitable for binding biological structures.

While the screening of biodiverse libraries has proven valuable, such libraries tend to be biased towards the frequency with which a particular organism is found in the native environment and may not necessarily represent the true population of the biodiversity found in a particular biological sample.

Because the libraries mentioned in that patent are generated from environmental samples for which little would be known about the genomic constitution of the library the procedure employs complicated normalisation methods to normalise the genomic constitution of the libraries.

While that procedure permits some normalisation of the genomes in an environmental sample, the methods that it describes are complicated, there is a risk that rare genomic DNA's will be lost when the methods are applied and / or that new biases will be introduced by the procedure.

Furthermore, because no information is known about the genomic constitution of the environmental sample, only limited bioinformatic data can be derived from a screen of the library.

There are, however, currently no available methods for screening normalised biodiverse peptide domain libraries in vivo wherein the entire composition and complexity of the library can be accurately estimated and wherein the screening process provides such comprehensive bioinformatic data useful for rational drug design.

Moreover, no methods have been described which are specifically designed for the construction of natural genomic sequence libraries that have been optimised for the expression of domains per se, rather than entire polypeptides.