Generation of diversity in combinatorial libraries

Details Generation of diversity in combinatorial libraries Generation of diversity in combinatorial libraries

2001-07-24

2003-10-28

Horlick, Kenneth R. (Department: 1637)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Biological or biochemical

C530S300000, C536S023100

Reexamination Certificate

active

06640192

ABSTRACT:

Biotech evolutionary methods, including combinatorial libraries and phage-display technology (PARMLEY & SMITH 1988; SCOTT & SMITH 1990; SMITH 1993), are used in the search for novel ligands of diagnostic, biomedical and pharmaceutical use (reviews; CORTESE 1996; COLLINS 1997). These methods, which use empirical procedures to select molecules with required characteristics, e.g. binding properties, from large populations of variant gene products has been compared to the process of natural evolution. Evolution includes the generation of mutation, selection of functionality over a time period and the ability of the systems to self-replicate. In particular natural systems use recombination to reassort mutations accumulated in the selected population to exponentially increase the combinations of mutations and thus increase the number of variants in the population. This latter aspect, namely the introduction of recombination within mutant genes has only recently been applied to biotech evolutionary methods, although it has been used to increase the size of initial phage-display libraries (e.g. WATERHOUSE 1993; TSURUSHITA 1996; SODOYER 1994; FISCH 1996). STEMMER 1994a, 1994b and 1995 teach that recombination amongst a population of DNA molecules can be achieved in vitro by PCR amplification of a mixture of small overlapping fragments with (1994a, 1994b) or without (STEMMER 1995) primer oligonucleotide sequences being used to drive the PCR reaction. The method is not applicable to recombination within a fully randomized (highly mutated) sequence since the method relies on high homology of the overlapping sequences at the site of recombination. STEMMER 1994b and CRAMERI 1996a do, however, demonstrate the usefulness of in vitro recombination for molecular evolution, where CRAMERI 1996b also demonstrate the use of the method in conjunction with phage-display, even though their method is confined to regions of low mutant density (ca. 0.5-1% of the bases are mutated in their method) as they state “the advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution” (STEMMER 1994b). We point out that this is due to the self-evident fact that the number of variants created by mutagenesis introducing base changes in existing mutant structures is an additive i.e., a linearly increasing function, whereas the use of recombination between mutated variants yields novel variants as an exponential function of the initial number of variants. The classical phage-display libraries are thus at a grave disadvantage for the generation of novel variants; e.g. to encompass all the possible variants of an octapeptide sequence 20
8
=2.56×10
10
different variants would be required.
MARKS 1992 state the importance of recombination in the generation of higher specificity in combinatorial libraries e.g. in attaining antibodies of higher specificity and binding constants in the form of reshuffling light and heavy chains of immunoglobulins displayed in phage-display libraries. These authors do not instruct how the shuffling of all the light and heavy chains in a population heterogeneous in. both chains can be achieved, e.g. by a vector allowing recombination. Heavy and light chains were selected one after the other, i.e. an optimal heavy chain first selected from a heterogeneous heavy chain population in the presence of a constant light chain, then by preparing a new library, an optimal light chain in combination with the preselected optimal heavy chain. The extensive time consuming sequential optimization strategies currently utilized including consensus-mutational libraries, in vivo mutagenesis, error-pone PCR as well as chain shuffling are summarized in
FIGS. 5 and 6
of COLLINS 1997.
General Background to Phase and Phape-display Libraries
Gene libraries are generated containing extremely large number (10
6
to 10
10
) of variants. The variant gene segments are fused to a coat protein gene of a filamentous bacteriophage (e.g. M13, fd or fl), and the fusion gene is inserted into the genome of the phage or of a phagemid. A phagemid is defined as a plasmid containing the packaging and replication origin of the filamentous bacteriophage. This latter property allows the packaging of the phagemid genome into a phage coat when it is present in an
Escherichia coli
host strain infected with a filamentous phage (superinfection). The packaged particles produced, be they phage or phagemid, display the fusion protein on the surface of the particles secreted into the medium. Such packaged particles are able to inject their genomes into a new host bacterium, where they can be propagated as phage or plasmids, respectively. The special property of the system lies in the fact that since the packaging takes place in individual cells usually infected by a single variant phage/phagemid, the particles produced on propogation contain the gene encoding the particular variant displayed on the particle's surface. Several cycles of affinity selection for clones exhibiting the required properties due to the particular property of the variant protein displayed, e.g. binding to a particular target molecule immobilized on a surface, followed by amplification of the enriched clones leads to the isolation of a small number of different clones having these properties. The primary structure of these variants can then be rapidly elucidated by sequencing the hypermutated segment of the variant gene.
Efficiency of Producing Combinatorial Libraries
There are a number of factors which limit the potential of this technology. The first is the number and diversity of the variants which can be generated in the primary library. Most libraries have been generated by transformation of ligated DNA preparations into
Escherichia coli
by electroporation. This gives an efficiency of ca. 0.1 to 1×106 recombinants/microgram ligated phage DNA. The highest cloning efficiency reported (of 107 recombinants per microgram insert DNA) is obtained using special lambda vectors into which a single filamentous phage vector is inserted, in a special cloning site, bracketted by a duplication of the filamentous phage replication/packaging origin (AMBERG 1993; HOGREFE 1993a+b). The DNA construct is efficiently introduced into the
Escherichia coli
host after packaging into a lambda bacteriophage coat in an in vitro lambda packaging mix. Infection of a strain carrying such a hybrid phagemid by an M13-helper phage allows excision and secretion of the insert packed in a filamentous phage coat. Neither AMBERG 1993 nor HOGREFE 1993a+b instruct on how the method may be used to introduce recombination during this procedure. Although they mention that the efficiency may be improved by the use of type IIS restriction endonucleases during the construction of the concatemers used as substrate for the in vitro packaging no examples are given and in the ensuing five years no examples have appeared in the literature. The procedure described in our invention also uses the high efficiency of the in vitro lambda packaging, but maximizes the capacity of the cloning vector by using a cosmid vector (8) in which many copies (say 8) of the phagemid are inserted in each construct. One of the surprising innovative aspects of this procedure is the discovery of a number of protocols for the de novo synthesis of large hypervariable libraries. One type is particularly efficient, in that phagemid/cosmid vectors are forced to integrate into the hybrid concatamers oriented in the same orientation. Any variant of the protocol which does not ensure this feature does not work efficiently.
The Use of Type IIS Restriction Endonucleases
SZYBALSKI 1991 teaches a large number of novel applications for type IIS restriction endonucleases, including precise trimming of DNA, retrieval of cloned DNA, gene assembly, use as a universal restriction enzyme, cleavage of single-stranded DNA, detection of point mutations, tandem amplification, printing amplification reactions and localization of methylated bases. They d

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