WO2002022810A2 - Criblage combinatoire de populations mixtes d'organismes - Google Patents

Criblage combinatoire de populations mixtes d'organismes Download PDF

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Publication number
WO2002022810A2
WO2002022810A2 PCT/US2001/029712 US0129712W WO0222810A2 WO 2002022810 A2 WO2002022810 A2 WO 2002022810A2 US 0129712 W US0129712 W US 0129712W WO 0222810 A2 WO0222810 A2 WO 0222810A2
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biomolecule
bioactivity
nucleic acid
library
interest
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PCT/US2001/029712
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English (en)
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WO2002022810A3 (fr
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Jay M. Short
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Diversa Corporation
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Priority claimed from US09/663,620 external-priority patent/US7018793B1/en
Application filed by Diversa Corporation filed Critical Diversa Corporation
Priority to CA002391626A priority Critical patent/CA2391626A1/fr
Priority to JP2002527252A priority patent/JP2004524811A/ja
Priority to EP01971309A priority patent/EP1319068A2/fr
Priority to AU91208/01A priority patent/AU9120801A/en
Publication of WO2002022810A2 publication Critical patent/WO2002022810A2/fr
Publication of WO2002022810A3 publication Critical patent/WO2002022810A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • the present invention relates generally to screening and identification of bioactive molecules and more specifically to the production and screening of gene libraries generated from nucleic acid isolated from more than one organism for bioactive molecules or bioactivities.
  • bioactive compounds are derived from soil microorganisms. Many microbes inhabiting soils and other complex ecological communities produce a variety of compounds that increase their ability to survive and proliferate. These compounds " are generally thought to be nonessential for growth of the organism and are synthesized with the aid of genes involved in intermediary metabolism. Such secondary metabolites that influence the growth or survival of other organisms are known as "bioactive" compounds and serve as key components of the chemical defense arsenal of both micro- and macroorganisms. Humans have exploited these compounds for use as antibiotics, antiinfectives and other bioactive compounds with activity against a broad range of prokaryotic and eukaryotic pathogens (Barnes et al, Proc.Nat. Acad. Sci. U.S.A., 91, 1994).
  • a central core of modern biology is that genetic information resides in a nucleic acid genome, and that the information embodied in such a genome (i. e. , the genotype) directs cell function. This occurs through the expression of various genes in the genome of an organism and regulation of the expression of such genes.
  • the expression of genes in a cell or organism defines the cell or organism's physical characteristics (i.e., its phenotype). This is accomplished through the translation of genes into proteins. Determining the biological activity of a protein obtained from an environmental sample can provide valuable information about the role of proteins in the environments. In addition, such information can help in the development of biologies, diagnostics, therapeutics, and compositions for industrial applications.
  • the present invention provides methods to access this untapped biodiversity and to rapidly screen for sequences and activities of interest utilizing recombinant DNA technology.
  • This invention combines the benefits associated with the ability to rapidly screen natural compounds with the flexibility and reproducibility afforded with working with the genetic material of organisms.
  • Samples can be derived from a wide range of sources and include, for example, environmental libraries, samples containing more than one organisms (e.g., mixed populations of organisms), samples from unculturable organisms, deep sea vents and the like. As described herein, such samples provide a rich source of untapped molecules useful in biologies, therapeutics and industrial application, which prior to the present invention required laborious and time consuming methods for characterization and identification or were unable to be identified or characterized.
  • the invention provides a method for obtaining a bioactivity or a biomolecule of interest by screening a library of clones generated from nucleic acids from a mixed population of cells, for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule; and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is indicative of an effect of sequence variegation, thereby providing the bioactivity or biomolecule of interest.
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest by screening a library of clones generated from pooled nucleic acids obtained from a plurality of isolates for a specified bioactivity or biomolecule; and identifying a clone which contains the specified bioactivity or biomolecule.
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest.
  • the method includes screening a library of clones generated from pooled nucleic acids obtained from a plurality of isolates for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is indicative of an effect of introducing at least one sequence variegation, thereby providing the bioactivity or biomolecule of interest.
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest, wherein the method includes screening a library of clones generated from pooling individual gene libraries generated from the nucleic acids obtained from each of a plurality of isolates for a specified bioactivity or biomolecule and identifying a clone which contains the specified bioactivity or biomolecule.
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest by screening a library for a specified bioactivity or biomolecule wherein the library is generated from pooling individual gene libraries generated from the nucleic acids obtained from each of a plurality of isolates, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is indicative of an effect of introducing at least one sequence variegation, thereby providing the bioactivity or biomolecule of interest.
  • the invention provides a method of identifying a bioactivity or biomolecule of interest, including screening a library of clones generated from the nucleic acids from an enriched population of organisms for a specified bioactivity or biomolecule and identifying a clone containing the specified bioactivity or biomolecule.
  • the invention provides a method of identifying a bioactivity or biomolecule of interest by screening a library of clones generated from nucleic acids from an enriched population of organisms for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is indicative of an effect of introducing at least one sequence variegation, thereby providing the bioactivity or biomolecule of interest.
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest.
  • the bioactivity or biomolecule of interest is identified by incubating nucleic acids from a mixed population of organisms with at least one oligonucleotide probe having a detectable molecule and at least a portion of a nucleic acid sequence encoding a molecule of interest under conditions to allow interaction of complementary sequences, identifying nucleic acid sequences having a complement to the oligonucleotide probe using an analyzer that detects the detectable molecule.
  • a library is then generated from the identified nucleic acid sequences and the library is screened for a specified bioactivity or biomolecule.
  • Nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule is variegated and the variegated bioactivity or biomolecule compared with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is indicative of an effect of introducing at least one sequence variation, thereby providing the bioactivity or biomolecule of interest
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest by co-encapsulating in a microenvironment nucleic acids obtained from a mixed population of organisms, with at least one oligonucleotide probe having a detectable molecule and at least a portion of a nucleic acid sequence encoding a molecule of interest under such conditions and for such time as to allow interaction of complementary sequences, identifying encapsulated nucleic acids containing a complement to the oligonucleotide probe encoding the molecule of interest by separating the encapsulated nucleic acids with an analyzer that detects the detectable molecule, generating a library from the separated encapsulated nucleic acids, screening the library for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity
  • the invention provides a method including co- encapsulating in a microenvironment nucleic acids obtained from an isolate of a mixed population of organisms, with at least one oligonucleotide probe having a detectable marker and at least a portion of a polynucleotide sequence encoding a molecule having a bioactivity of interest under conditions and for such time as to allow interaction of complementary sequences, identifying encapsulated nucleic acids containing a complement to the oligonucleotide probe encoding the molecule of interest by separating the encapsulated nucleic acids with an analyzer that detects the detectable marker, generating a library from the separated encapsulated nucleic acids, screening the library for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity or biomolecule wherein a difference in the bioactivity or biomolecule is
  • the invention provides a method for obtaining a bioactivity or a biomolecule of interest by co-encapsulating in a microenvironment nucleic acids obtained from one or more isolates of a mixed population of organisms, with at least one oligonucleotide probe having a detectable marker and at least a portion of a polynucleotide sequence encoding a molecule having a bioactivity of interest under such conditions and for such time as to allow interaction of complementary sequences, identifying encapsulated nucleic acids containing a complement to the oligonucleotide probe encoding the molecule of interest by separating the encapsulated nucleic acids with an analyzer that detects the detectable marker, generating a library from the separated encapsulated nucleic acids, screening the library for a specified bioactivity or biomolecule, variegating a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or biomolecule with the specified bioactivity
  • the invention provides a method for identifying a bioactivity or a biomolecule of interest.
  • the method includes co-encapsulating in a microenvironment nucleic acids obtained from a mixture of isolates of a mixed population of organisms, with at least one oligonucleotide probe having a detectable marker and at least a portion of a polynucleotide sequence encoding a molecule having a bioactivity of interest under such conditions and for such time as to allow interaction of complementary sequences, identifying encapsulated nucleic acids containing a complement to the oligonucleotide probe encoding the molecule of interest by separating the encapsulated nucleic acids with an analyzer that detects the detectable marker, generating a library from the separated encapsulated nucleic acids, screening the library for a specified bioactivity or biomolecule, variegating the a nucleic acid sequence contained in a clone having the specified bioactivity or biomolecule, and comparing the variegated bioactivity or bio
  • Figure 1 is an illustration of the preferred embodiments of site-saturation mutagenesis.
  • Figure 2 shows an illustration of the gGeneration of an exhaustive set of chimeric combinations by synthetic ligation reassembly.
  • Figure 3 A and 3B are diagrams of the application of end-selection using a topoisomerase-guided selection step to select for desirable polynucleotides (e.g. the selection of full length molecules) generated by polynucleotide reassembly (e.g. by synthetic ligation reassembly).
  • desirable polynucleotides e.g. the selection of full length molecules
  • polynucleotide reassembly e.g. by synthetic ligation reassembly
  • Figure 4 illustrates various aspects of the end-selection technology.
  • Figure 5 illustrates a specific aspect of the end-selection technology where step of "c) Performing directed expression cloning" is achieved using the enzyme N.BstNBI in order to introduce "sticky ends" into an originally blunt-ended DNA in order to achieve directional expression cloning of the original blunt-ended DNA.
  • Figure 6 shows the activity of the enzyme exonuclease III.
  • the asterisk indicates that the enzyme acts from the 3' direction towards the 5' direction of the polynucleotide substrate.
  • Figure 7 shows an exemplary application of the enzyme exonuclease III in exonuclease-mediated polynucleotide reassembly.
  • Figure 8 shows the generation of a poly-binding nucleic acid strand.
  • Figure 9 shows the use of exonuclease treatment as a means to liberate 3' and 5 ' -terminal nucleotides from the unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but hybridized end to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated end. Shown also is the combined use of in vivo "repair”.
  • Figure 10 shows the use of exonuclease-mediated nucleic acid reassembly in an example in which one methylated poly-binding nucleic acid strand is annealed to several unmethylated mono-binding nucleic acid strands.
  • Figure 11 shows an illustration of the use of exonuclease-mediated nucleic acid reassembly in an example in which a plurality of methylated poly-binding nucleic acid strand are annealed to several unmethylated mono-binding nucleic acid strands.
  • Figure 12 shows a graph of the thermal stability of wild type (20F12) and mutant (Dhla 5) dehalogenase at 55°C.
  • Figure 13 shows a graph of the thermal stability of Dhla 5 dehalogenase at 55°C.
  • Figure 14 shows enzyme progress curves for Dhla 5 and 20F12 at 30°C and 55°C.
  • Figure 15 shows thermal stability of Dhla 5 dehalogenase at 80°C.
  • Figure 16 shows thermal stability of Dhla 5 and Dhla 8 dehalogenase at 80°C.
  • Figures 17 A, B and C show differential scanning calorimetry of the three dehalogenase variants. The data were recorded using a Perkin Elmer Pyris 1 at a scan rate of 10°C/min.
  • the present invention provides rapid screening of libraries derived from more than one organism, such as a mixed population of organisms from, for example, an environmental sample or an uncultivated population of organisms or a cultivated population of organisms.
  • gene libraries are generated by obtaining nucleic acids from a mixed population of organisms and cloning the nucleic acids into a suitable vector for transforming a plurality of clones to generate a gene library.
  • the gene library thus contains gene or gene fragments present in organisms of the mixed population.
  • the gene library can be an expression library, in which case the library can be screened for an expressed polypeptide having a desired activity.
  • the gene library can be screened for sequences of interest by, for example, PCR or hybridization screening.
  • nucleic acids from isolates of a sample containing a mixed population of organism are pooled and the pooled nucleic acids are used to generate a gene library.
  • Isolates is meant that a particular species, genus, family, order, or class of organisms is obtained or derived from a sample having more than one organism or from a mixed population of organisms. Nucleic acids from these isolated populations can then be used to generate a gene library. Isolates can be obtained from by selectively filtering or culturing a sample containing more than one organism or a mixed population of organisms. For example, isolates of bacteria can be obtained by filtering the sample through a filter which excludes organisms based on size or by culturing the sample on media that allows from selective growth or selective inhibition of certain populations of organisms.
  • An "enriched population” is a population of organisms wherein the percentage of organisms belonging to a particular species, genus, family, order or class of organisms is increased with respect to the population as a whole.
  • selective growth or inhibition media can increase the overall number of organisms.
  • One can enrich for prokaryotic organisms with respect to the total number of organisms in the population.
  • a particular species, genus, family, order or class of organisms can be enriched by growing a mixed population on a selective media that inhibits or promotes the growth of a subpopulation within the mixed population.
  • nucleic acids from a plurality (e.g., two or more) of isolates from a mixed population of organisms are used to generate a plurality of gene libraries containing a plurality of clones, and the gene libraries from at least two isolates are then pooled to obtain a "pooled isolate library.”
  • Such screening techniques include, for example, contacting a clone, clonal population, or population of nucleic acid sequences with a substrate or substrates having a detectable molecule that provides a detectable signal upon interaction with the bioactivity or biomolecule of interest.
  • the substrate can be an enzymatic substrate, a bioactive molecule, an oligonucleotide, and the like.
  • gene libraries are generated, clones are either exposed to a chromagenic or fluorogenic substrate or substrate(s) of interest, or hybridized to a labeled probe (e.g., an oligonucleotide having a detectable molecule) having a sequence corresponding to a sequence of interest and positive clones are identified by a detectable signal (e.g., fluorescence emission).
  • a labeled probe e.g., an oligonucleotide having a detectable molecule
  • expression libraries generated from a mixed population of organisms are screened for an activity of interest. Specifically, expression libraries are generated, clones are exposed to the substrate or substrate(s) of interest, and positive clone are identified and isolated.
  • the present invention does not require cells to survive. The cells only need to be viable long enough to produce the molecule to be detected, and can thereafter be either viable or nonviable cells, so long as the expressed biomolecule (e.g., an enzyme) remains active.
  • the invention provides an approach that combines direct cloning of genes encoding novel or desired bioactivities from environmental samples with a high-throughput screening system designed for the rapid discovery of new molecules, for example, enzymes.
  • the approach is based on the construction of environmental "expression libraries" which can represent the collective genomes of numerous naturally occurring microorganisms archived in cloning vectors that can be propagated in E. coli or other suitable host cells. Because the cloned DNA can be initially extracted directly from environmental samples or from isolates of the environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture.
  • a normalization of the environmental DNA present in these samples could allows a more equal representation of the DNA from all of the species present in a sample. Normalization techniques (described below) can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample that may be under-represented by several orders of magnitude compared to the dominant species in the sample. Normilization can occur in any of the foregoing embodiments following obtaining nucleic acids from the sample or isolate(s).
  • the invention provides a high-throughput capillary array system for screening that allows one to assess an enormous number of clones to identify and recover cells encoding useful enzymes, as well as other biomolecules (e.g. , ligands).
  • the capillary array-based techniques described herein can be used to screen, identify and recover proteins having a desired bioactivity or other ligands having a desired binding affinity.
  • binding assays may be conducted by using an appropriate substrate or other marker that emits a detectable signal upon the occurrence of the desired binding event.
  • fluorescence activated cell sorting can be used to screen and isolate clones having an activity or sequence of interest.
  • FACS machines have been employed in the studies focused on the analyses of eukaryotic and prokaryotic cell lines and cell culture processes.
  • FACS has also been utilized to monitor production of foreign proteins .in both eukaryotes and prokaryotes to study, for example, differential gene expression, and the like.
  • the detection and counting capabilities of the FACS system have been applied in these examples.
  • FACS has never previously been employed in a discovery process to screen for and recover bioactivities in prokaryotes.
  • the present invention does not require cells to survive, as do previously described technologies, since the desired nucleic acid (recombinant clones) can be obtained from alive or dead cells.
  • the cells only need to be viable long enough to produce the compound to be detected, and can thereafter be either viable or non- viable cells so long as the expressed biomolecule remains active.
  • the present invention also solves problems that would have been associated with detection and sorting of E. coli expressing recombinant enzymes, and recovering encoding nucleic acids.
  • the present invention includes within its embodiments any apparatus capable of detecting fluorescent wavelengths associated with biological material, such apparati are defined herein as fluorescent analyzers (one example of which is a FACS apparatus).
  • nucleic acid sequences of interest can be identified or "biopanned" by contacting a clone, device (e.g. a gene chip), filter, or nucleic acid sample with a probe labeled with a detectable molecule.
  • the probe will typically have a sequence that is substantially identical to the nucleic acid sequence of interest, Alternatively, the probe will be a fragment or full length nucleic acid sequence encoding a polypeptide of interest.
  • the prove and nucleic acids are incubated under conditions and for such time as to allow the probe and a substantially complementary sequence to hybridize.
  • Hybridization stringency will vary depending on, for example, the length and GC content of the probe. Such factors can be determined empirically (See, for example, Sambrook et al. , Molecular Cloning —A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, M. Ausubel et ah, eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., most recent Supplement)).
  • the complementary sequence can be PCR amplified, identified by hybridization techniques (e.g., exposing the probe and nucleic acid mixture to a film), or detecting the nucleic acid using a chip.
  • the present invention allows the rapid screening of complex environmental libraries, containing, for example, genomic sequences from thousands of different organisms or subsets and isolates thereof.
  • the benefits of the present invention can be seen, for example, in screening a complex environmental sample. Screening of a complex sample previously required one to use labor intensive methods to screen several million clones to cover the genomic biodiversity.
  • the invention represents an extremely high-throughput screening method which allows one to assess this enormous number of clones.
  • the method disclosed allows the screening anywhere from about 30 million to about 200 million clones per hour for a desired nucleic acid sequence, biological activity, or biomolecule of interest. This allows the thorough screening of environmental libraries for clones expressing novel bioactivities or biomolecules.
  • sequence or bioactivity of interest e.g., an enzyme of interest
  • the sequence or polynucleotide encoding the bioactivity of interest can be evolved, mutated or derived to modify the amino acid sequence to provide, for example, modified activities such as increased thermostability, specificity or activity.
  • amino acid is a molecule having the structure wherein a central carbon atom (the ⁇ -carbon atom) is linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a “carboxyl carbon atom”), an amino group (the nitrogen atom of which is referred to herein as an "amino nitrogen atom"), and a side chain group, R.
  • an amino acid loses one or more atoms of its amino acid carboxylic groups in the dehydration reaction that links one amino acid to another.
  • an amino acid is referred to as an "amino acid residue."
  • Protein or “polypeptide” refers to any polymer of two or more individual amino acids (whether or not naturally occurring) linked via a peptide bond, and occurs when the carboxyl carbon atom of the carboxylic acid group bonded to the ⁇ -carbon of one amino acid (or amino acid residue) becomes covalently bound to the a ino nitrogen atom of amino group bonded to the ⁇ -carbon of an adjacent amino acid.
  • protein is understood to include the terms “polypeptide” and “peptide” (which, at times may be used interchangeably herein) within its meaning.
  • proteins comprising multiple polypeptide subunits (e.g., DNA polymerase III, RNA polymerase II) or other components (for example, an RNA molecule, as occurs in telomerase) will also be understood to be included within the meaning of "protein” as used herein.
  • proteins comprising multiple polypeptide subunits (e.g., DNA polymerase III, RNA polymerase II) or other components (for example, an RNA molecule, as occurs in telomerase) will also be understood to be included within the meaning of "protein” as used herein.
  • fragments of proteins and polypeptides are also within the scope of the invention and may be referred to herein as "proteins.”
  • a particular amino acid sequence of a given protein (z. e. , the polypeptide' s
  • primary structure when written from the amino-terminus to carboxy-terminus is determined by the nucleotide sequence of the coding portion of a mRNA, which is in turn specified by genetic information, typically genomic DNA (including organelle DNA, e.g., mitochondrial or chloroplast DNA).
  • genomic DNA including organelle DNA, e.g., mitochondrial or chloroplast DNA.
  • isolated or purified when referring to a nucleic acid sequence or a polypeptide sequence, respectively, means altered “by the hand of man” from its natural state; . e. , if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polynucleotide or a polypeptide naturally present in a living animal a biological sample or an environmental sample in its natural state is not “isolated” or “purified”, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated” or “purified”, as the term is employed herein.
  • polynucleotides when introduced into host cells in culture or in whole organisms, still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • the polynucleotides and polypeptides may occur in a composition, such as a media formulation (solutions for introduction of polynucleotides or polypeptides, for example, into cells or compositions or solutions for chemical or enzymatic reactions).
  • Polynucleotide or “nucleic acid sequence” refers to a polymeric form of nucleotides. In some instances a polynucleotide refers to a sequence that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • a polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • polynucleotide encompasses genomic DNA or RNA (depending upon the organism, i.e., RNA genome of viruses), as well as mRNA encoded by the genomic DNA, and cDNA.
  • enzymes are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, selectivity, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries. Enzyme-based processes have been gradually replacing many conventional chemical-based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes.
  • Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far identified have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments.
  • such enzymes must function at temperatures above 100°C in terrestrial hot springs and deep sea thermal vents, at temperatures below 0°C in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge.
  • the invention provides not only a source of materials for the development of biologies, therapeutics, and enzymes for industrial applications, but also provides a new materials for further processing by, for example, directed evolution and mutagenesis to develop molecules or polypeptides modified for particular activity, specificity or conditions.
  • the invention can be used to obtain and identify polynucleotides and related sequence specific information from, for example, infectious microorganisms present in the environment such as, for example, in the gut of various macroorganisms.
  • Identifying novel enzymes in an environmental sample is one solution to this problem.
  • the invention provides methods, compositions and sources for the development of biologies, diagnostics, therapeutics, and compositions for industrial applications.
  • the methods and compositions of the invention provide for the identification of lead drug compounds present in an environmental sample.
  • the methods of the invention provide the ability to mine the environment for novel drugs or identify related drugs contained in different microorganisms.
  • lead compounds drug candidates
  • natural product collections synthetic chemical collections
  • synthetic combinatorial chemical libraries such as nucleotides, peptides, or other polymeric molecules that have been identified or developed as a result of environmental mining.
  • Each of these sources has advantages and disadvantages.
  • the success of programs to screen these candidates depends largely on the number of compounds entering the programs, and pharmaceutical companies have to date screened hundred of thousands of synthetic and natural compounds in search of lead compounds. Unfortunately, the ratio of novel to previously-discovered compounds has diminished with time.
  • the discovery rate of novel lead compounds has not kept pace with demand despite the best efforts of pharmaceutical companies. There exists a strong need for accessing new sources of potential drug candidates. Accordingly, the invention provides a rapid and efficient method to identify and characterize environmental samples that contain novel drug compounds.
  • bioactive compounds are derived from soil microorganisms. These compounds are generally thought to be nonessential for growth of the organism and are synthesized with the aid of genes involved in intermediary metabolism hence their name — “secondary metabolites”. Secondary metabolites that influence the growth or survival of other organisms are known as “bioactive” compounds and serve as key components of the chemical defense arsenal of both micro- and macro-organisms.
  • bioactive compounds of microbial origin Approximately 6,000 bioactive compounds of microbial origin have been characterized, with more than 60% produced by the gram positive soil bacteria of the genus Streptomyces. (Barnes et al, Proc.Nat. Acad. Sci. U.S.A., 91, 1994). Of these, at least 70 are currently used for biomedical and agricultural applications.
  • the largest class of bioactive compounds, the polyketides include a broad range of antibiotics, immunosuppressants and anticancer agents which together account for sales of over $5 billion per year.
  • the invention provides methods of identifying a nucleic acid sequence encoding a polypeptide having either known or unknown function. For example, much of the diversity in microbial genomes results from the rearrangement of gene clusters in the genome of microorganisms. These gene clusters can be present across species or phylogenetically related with other organisms.
  • genes are clustered, in structures referred to as "gene clusters," on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster.
  • the gene cluster, the promoter, and additional sequences that function in regulation altogether are referred to as an "operon" and can include up to 20 or more genes, usually from 2 to 6 genes.
  • a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.
  • Some gene families consist of identical members. Clustering is a prerequisite for maintaining identity between genes, although clustered genes are not necessarily identical. Gene clusters range from extremes where a duplication is generated to adjacent related genes to cases where hundreds of identical genes lie in a tandem array. Sometimes no significance is discernable in a repetition of a particular gene. A principal example of this is the expressed duplicate insulin genes in some species, whereas a single insulin gene is adequate in other mammalian species.
  • polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracyclines and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin). Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents.
  • Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in length and patterns of functionality and cyclization. Polyketide synthase genes fall into gene clusters and at least one type (designated type I) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins. Other types of proteins that are the product(s) of gene clusters are also contemplated, including, for example, antibiotics, antivirals, antitumor agents and regulatory proteins, such as insulin.
  • the ability to select and combine desired components from a library of polyketides and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing.
  • the method(s) of the present invention make it possible to, and facilitate the cloning of, novel polyketide synthases and other gene clusters, since one can generate gene banks with clones containing large inserts (especially when using the f-factor based vectors), which facilitates cloning of gene clusters.
  • a gene cluster can be ligated into a vector containing an expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related array activity from the ligated gene clusters.
  • Use of vectors which have an exceptionally large capacity for exogenous nucleic acid introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E. coli.
  • This f-factor of E. coli is a plasmid which affects high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large nucleic acid fragments, such as gene clusters from mixed microbial samples.
  • nucleic acid isolated or derived from these samples can be inserted into a vector or a plasmid prior to screening of the polynucleotides.
  • vectors or plasmids are typically those containing expression regulatory sequences, including promoters, enhancers and the like.
  • the invention provides novel systems to clone and screen mixed populations of organisms enriched samples, or isolates thereof for polynucleotides encoding molecules having an activity of interest, enzymatic activities and bioactivities of interest in vitro.
  • the method(s) of the invention allow the cloning and discovery of novel bioactive molecules in vitro, and in particular novel bioactive molecules derived from uncultivated or cultivated samples. Large size gene clusters, genes and gene fragments can be cloned, sequenced and screened using the method(s) of the invention.
  • the method(s) of the invention allow one to clone screen and identify polynucleotides and the polypeptides encoded by these polynucleotides in vitro from a wide range of environmental samples .
  • the invention allows one to screen for and identify polynucleotide sequences from complex environmental samples, enriched samples thereof, or isolates thereof.
  • Gene libraries can be generated from cell free samples, so long as the sample contains nucleic acid sequences, or from samples containing cells, cellular material or viral particles.
  • the organisms from which the libraries may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, lower eukaryotic microorganisms such as fungi, algae and protozoa, as well as mixed populations of plants, plant spores and pollen.
  • the organisms may be cultured organisms or uncultured organisms obtained from environmental samples and includes extremophiles, such as thermophiles, hyperthermophiles, psychrophiles and psychrotrophs.
  • Sources of nucleic acids used to generate a DNA library can be obtained from environmental samples, such as, but not limited to, microbial samples obtained from Arctic and Antarctic ice, water or permafrost sources, materials of volcanic origin, materials from soil or plant sources in tropical areas, droppings from various organisms including mammals and invertebrates, as well as dead and decaying matter and the like.
  • the nucleic acids used to generate the gene libraries can be obtained, for example, from enriched subpopulations or ioslates of the sample.
  • DNA of a plurality of isolates can be pooled to create a source of nucleic acids for generation of the library.
  • the nucleic acids can be obtained from a plurality of isolates, a plurality of gene libraries generated from the plurality of isolates to obtain a plurality of gene libraries. Two or more of the gene libraries can be pooled or combined to obtain a pooled isolate library.
  • nucleic acids may be recovered from either a cultured or non-cultured organism and used to produce an appropriate gene library (e.g., a recombinant expression library) for subsequent determination of the identity of the particular biomolecule of interest (e.g., a polynucleotide sequence) or screened for a bioactivity of interest (e.g., an enzyme or biological activity).
  • an appropriate gene library e.g., a recombinant expression library
  • an environmental sample is any sample containing organisms or polynucleotides or a combination thereof.
  • an environmental sample can be obtained from any number of sources (as described above), including, for example, insect feces, hot springs, soil and the like. Any source of nucleic acids in purified or non-purified form can be utilized as starting material. Thus, the nucleic acids may be obtained from any source which is contaminated by an organism or from any sample containing cells.
  • the environmental sample can be an extract from any bodily sample such as blood, urine, spinal fluid, tissue, vaginal swab, stool, amniotic fluid or buccal mouthwash from any mammalian organism.
  • sample can be a tissue sample, salivary sample, fecal material or material in the digestive tract of the organism.
  • An environmental sample also includes samples obtained from extreme environments including, for example, hot sulfur pools, volcanic vents, and frozen tundra. The sample can come from a variety of sources.
  • the sample in horticulture and agricultural testing can be a plant, fertilizer, soil, liquid or other horticultural or agricultural product; in food testing the sample can be fresh food or processed food (for example infant formula, seafood, fresh produce and packaged food); and in environmental testing the sample can be liquid, soil, sewage treatment, sludge and any other sample in the environment which is considered or suspected of containing an organism or polynucleotides.
  • the sample When the sample is a mixture of material containing a mixed population of organisms, for example, blood, soil or sludge, it can be treated with an appropriate reagent which is effective to open the cells and expose or separate the strands of nucleic acids. Although not necessary, this lysing and nucleic acid denaturing step will allow cloning, amplification or sequencing to occur more readily. Further, if desired, the mixed population can be cultured prior to analysis in order to purify or enrich a particular population or a desired isolate (e.g., an isolate of a particular species, genus, or family of organisms) and thus obtaining a purer sample. This is not necessary, however.
  • a desired isolate e.g., an isolate of a particular species, genus, or family of organisms
  • cultiiring of organisms in the sample can include culturing the organisms in microdroplets and separating the cultured microdroplets with a cell sorter into individual wells of a multi-well tissue culture plate.
  • the sample can be cultured on any number of selective media compositions designed to inhibit or promote growth of a particular subpopulation of organisms.
  • nucleic acids can be obtained from the isolates as described below. The nucleic acids obtained from the isolates can be used to generate a gene library or, alternatively, be pooled with other isolate fractions of the sample wherein the pooled nucleic acids are used to generate a gene library.
  • the isolates can be cultured prior to extraction of nucleic acids or can be uncultured. Methods of isolating specific populations of organisms present in a mixed population
  • the sample comprises nucleic acids from, for example, a diverse and mixed population of organisms (e.g., microorganisms present in the gut of an insect). Nucleic acids are isolated from the sample using any number of methods for DNA and RNA isolation. Such nucleic acid isolation methods are commonly performed in the art. Where the nucleic acid is RNA, the RNA can be reversed transcribed to DNA using primers known in the art. Where the DNA is genomic DNA, the DNA can be sheared using, for example, a 25 gauge needle.
  • the nucleic acids can be cloned into an appropriate vector.
  • the vector used will depend upon whether the DNA is to be expressed, amplified, sequenced or manipulated in any number of ways known in the art (see, for example, U.S. Patent No. 6,022,716 which discloses high throughput sequencing vectors). Cloning techniques are known in the art or can be developed by one skilled in the art, without undue experimentation. The choice of a vector will also depend on the size of the polynucleotide sequence and the host cell to be employed in the methods of the invention.
  • the vector used in the invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retro viruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g-., coat protein, spike glycoprotein, capsid protein).
  • viruses e.g., retro viruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like
  • selected portions thereof e.g-., coat protein, spike glycoprotein, capsid protein.
  • cosmids and phagemids are typically used where the specific nucleic acid sequence to be analyzed or modified is large because these vectors are able to stably propagate large polynucleotides.
  • the vector containing the cloned nucleic acid sequence can then be amplified by plating (t.e., clonal amplification) or transfecting a suitable host cell with the vector (e.g., a phage on an E. coli host).
  • the cloned nucleic acid sequence is used to prepare a library for screening (e.g., expression screening, PCR screening, hybridization screening or the like) by transforming a suitable organism.
  • Hosts known in the art are transformed by artificial introduction of the vectors containing the nucleic acid sequence by inoculation under conditions conducive for such transformation.
  • transform or transformation is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (e.g., DNA exogenous to the cell).
  • new DNA e.g., DNA exogenous to the cell
  • a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • a transformed cell or host cell generally refers to a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule not normally present in the host organism.
  • a particularly type of vector for use in the invention contains an f-factor origin replication.
  • the f-factor (or fertility factor) in E. coli is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself.
  • cloning vectors referred to as "fosmids" or bacterial artificial chromosome (BAG) vectors are used. These are derived from E. coli f-factor which is able to stably integrate large segments of DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable environmental gene library.
  • the nucleic acids derived from a mixed population or sample may be inserted into the vector by a variety of procedures.
  • the nucleic acid sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • a typical cloning scenario may have DNA "blunted" with an appropriate nuclease (e.g., Mung Bean Nuclease), methylated with, for example, EcoR /Methylase and ligated to EcoR I tinkers GGAATTCC (S ⁇ Q ID NO: 1).
  • the linkers are then digested with an EcoR /Restriction Endonuclease and the DNA size fractionated (e.g., using a sucrose gradient).
  • the resulting size fractionated DNA is then ligated into a suitable vector for sequencing, screening or expression (e.g., a lambda vector and packaged using an in vitro lambda packaging extract).
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method by procedures well known in the art.
  • CaCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • DNA include calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, as well as others known in the art, may be used.
  • Eukaryotic cells can also be cotransfected with a second foreign DNA molecule encoding a selectable marker, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • the eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae), an insect cell (e.g., Drosophila sp.) or may be a mammalian cell, including a human cell.
  • yeast cell e.g., Saccharomyces cerevisiae
  • insect cell e.g., Drosophila sp.
  • mammalian cell including a human cell.
  • Eukaryotic systems and mammalian expression systems, allow for post- translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for processing of the primary transcript, glycosylation, phosphorylation, or secretion of the gene product should be used.
  • host cell lines may include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.
  • the clones are resuspended in a liquid media, for example, a nutrient rich broth or other growth media known in the art.
  • a liquid media for example, a nutrient rich broth or other growth media known in the art.
  • the media is a liquid media which can be readily pipetted.
  • One or more media types containing at least one clone of the library is then introduced either individually or together as a mixture, into capillaries (all or a portion thereof) in a capillary array.
  • the library is first biopanned prior to introduction or delivery into a capillary device or other screening techniques.
  • biopanning methods enrich the library for sequences or activities of interest. Examples of methods for biopanning or enrichment are described below.
  • the library can be screened or sorted to enrich for clones containing a sequence or activity of interested based on polynucleotide sequences present in the library or clone.
  • the invention provides methods and compositions useful in screening organisms for a desired biological activity or biological sequence and to assist in obtaining sequences of interest that can further be used in directed evolution, molecular biology, biotechnology and industrial applications.
  • the invention provides methods to rapidly screen, enrich and/or identify sequences in a sample by screening and identifying the nucleic acid sequences present in the sample.
  • the invention increases the repertoire of available sequences that can be used for the development of diagnostics, therapeutics or molecules for industrial applications. Accordingly, the methods of the invention can identify novel nucleic acid sequences encoding proteins or polypeptides having a desired biological activity.
  • biopanning refers to a process for identifying clones having a specified biological activity by screening for sequence homology in a library of clones.
  • the probe sequence used for selectively interacting with the target sequence of interest in the library can be a full-length coding region sequence or a partial coding region sequence for a known bioactivity.
  • the library can be probed using mixtures of probes comprising at least a portion of the sequence encoding a known bioactivity or having a desired bioactivity.
  • These probes or probe libraries are preferably single- stranded.
  • the library is preferably been converted into single- stranded form.
  • the probes that are particularly suitable are those derived from DNA encoding bioactivities having an activity similar or identical to the specified bioactivity which is to be screened.
  • the probes can be used to PCR amplify and thus select target sequences.
  • the probe sequences can be used as hybridization probes which can be used to identify sequences with substantial or a desired homology.
  • in vivo biopanning may be performed utilizing a FACS-based machine, gene libraries or expression libraries are constructed with vectors which contain elements which stabilize transcribed RNA.
  • the inclusion of sequences which result in secondary structures such as hairpins which are designed to flank the transcribed regions of the RNA would serve to enhance their stability, thus increasing their half life within the cell.
  • the probe molecules used in the biopanning process consist of oligonucleotides labeled with detectable molecules that provide a detectable signal upon interaction with a target sequence (e.g., only fluoresce upon binding of the probe to a target molecule).
  • Various dyes or stains can be used to intercalate or associate with nucleic acid in order to "label" the oligonucleotides.
  • These probes are introduced into the recombinant cells of the library using one of several transformation methods.
  • the probe molecules interact or hybridize to the transcribed target mRNA or DNA resulting in DNA/RNA heteroduplex molecules or DNA/DNA duplex molecules. Binding of the probe to a target will yield a detectable signal (e.g., a fluorescent signal) which is detected and sorted by a FACS machine, or the like, during the screening process.
  • the probe DNA should be at least about 10 bases and preferably at least 15 bases. In one embodiment, an entire coding region of one part of a pathway may be employed as a probe. Where the probe is hybridized to the target DNA in an in vitro system, conditions for the hybridization in which target DNA is selectively isolated by the use of at least one DNA probe will be designed to provide a hybridization stringency of at least about 50% sequence identity, more particularly a stringency providing for a sequence identity of at least about 70%.
  • Hybridization techniques for probing a microbial DNA library to isolate target DNA of potential interest are well known in the art and any of those which are described in the literature are suitable for use herein including, for example, chip-based assays, membrane-based assays, and the like.
  • Clones can then be further screened for clones which display an activity of interest.
  • Clones can be shuttled in alternative hosts for expression of active compounds, or screened using methods described herein.
  • An alternative to the in vivo biopanning described above is an encapsulation techniques such as, for example, gel microdroplets, which may be employed to localize multiple clones in one location to be screened on a FACS machine. Clones can then be broken out into individual clones to be screened again on a FACS machine to identify positive individual clones. Screening in this manner using a FACS machine is fully application Ser. No. 08/876,276 filed Jun. 16, 1997. Thus, for example, if a clone mixture has a desirable activity, then the individual clones may be recovered and rescreened utilizing a FACS machine to determine which of such clones has the specified desirable activity.
  • an encapsulation techniques such as, for example, gel microdroplets
  • encapsulation strategies and compounds or polymers can be used with the present invention.
  • high temperature agarose can be employed for making microdroplets stable at high temperatures, allowing stable encapsulation of cells subsequent to heat-kill steps utilized to remove all background activities when screening for thermostable bioactivities.
  • Encapsulation can be in beads, high temperature agaroses, gel microdroplets, cells, such as ghost red blood cells or macrophages, liposomes, or any other means of encapsulating and localizing molecules.
  • liposomes For example, methods of preparing liposomes have been described (e.g., U.S. Patent No.'s 5,653,996, 5393530 and 5,651,981), as well as the use of liposomes to encapsulate a variety of molecules (e.g., U.S. Patent Nos. 5,595,756, 5,605,703, 5,627,159, 5,652,225, 5,567,433, 4,235,871, 5,227,170). Entrapment of proteins, viruses, bacteria and DNA in erythrocytes during endocytosis has been described, as well (see, for example, Journal of Applied Biochemistry 4, 418-435 (1982)).
  • Erythrocytes employed as carriers in vitro or in vivo for substances entrapped during hypo-osmotic lysis or dielectric breakdown of the membrane have also been described (reviewed in Ihler, G. M. (1983) J. Pharm. Ther). These techniques are useful in the present invention to encapsulate samples in a microenvironment for screening.
  • Microenvironment is any molecular structure which provides an appropriate environment for facilitating the interactions necessary for the method of the invention.
  • An environment suitable for facilitating molecular interactions include, for example, liposomes.
  • Liposomes can be prepared from a variety of lipids including phospholipids, glycolipids, steroids, long-chain alkyl esters; e.g., alkyl phosphates, fatty acid esters; e.g., lecithin, fatty amines and the like.
  • a mixture of fatty material may be employed such a combination of neutral steroid, a charge amphiphile and a phospholipid.
  • Illustrative examples of phospholipids include lecithin, sphingomyelin and dipalmitoylphos-phatidylcholine.
  • Representative steroids include cholesterol, cholestanol and lanosterol.
  • Representative charged amphiphilic compounds generally contain from 12-30 carbon atoms.
  • a normalization step is performed prior to generation of the expression library, the expression library is then generated, the expression library so generated is then biopanned, and the biopanned expression library is then screened using a high throughput cell sorting and screening instrument.
  • a normalization step is performed prior to generation of the expression library, the expression library is then generated, the expression library so generated is then biopanned, and the biopanned expression library is then screened using a high throughput cell sorting and screening instrument.
  • the nucleic acids used to generate a library can be obtained, for example, from environmental samples, mixed populations of organisms (e.g., cultured or uncultured), enriched populations thereof, and isolates thereof.
  • the screening techniques include, for example, hybridization screening, PCR screening, expression screening, and the like.
  • the gel microdroplet technology has had significance in amplifying the signals available in flow cytometric analysis, and in perrnitting the screening of microbial strains in strain improvement programs for biotechnology.
  • Wittrup et al (Biotechnolo.Bioeng. (1993) 42:351-356) developed a microencapsulation selection method which allows the rapid and quantitative screening of >10 6 yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400-fold single-pass enrichment for high-secretion mutants.
  • Gel microdroplet or other related technologies can be used in the present invention to localize, sort as well as amplify signals in the high throughput screening of recombinant libraries. Cell viability during the screening is not an issue or concern since nucleic acid can be recovered from the microdroplet.
  • the enriched clones are suspended in a liquid media such as a nutrient broth or other growth media.
  • a liquid media such as a nutrient broth or other growth media.
  • the enriched clones comprise a plurality of host cells transformed with constructs comprising vectors into which have been incorporated nucleic acid sequences derived from a sample (e.g., mixed poulations of organisms, isolates thereof, and the like).
  • Liquid media containing a subset of clones and one or more substrates having a detectable molecule is then introduced or contacted, either individually or together as a mixture, with the enriched clones (e.g., into capillaries in a capillary array).
  • Interaction (including reaction) of the substrate and a clone expressing an enzyme having the desire enzyme activity produces a product or a detectable signal, which can be spatially detected to identify one or more clones or capillaries containing at least one signal-producing clone.
  • the signal-producing clones or nucleic acids contained in the signal-producing clone can then be recovered using any number of techniques.
  • a “substrate” as used herein includes, for example, substrates for the detection of a bioactivity or biomolecule (e.g., an enzymes and their specific enzyme activities). Such substrates are well known in the art. For example, various enzymes and suitable substrates specific for such enzymes are provided in Molecular Probes, Handbook Of Fluorescent Probes and Research Chemical (Molecular Probes, Inc.; Eugene, OR), the disclosure of which is incorporated herein by reference.
  • the substrate can have a detectable molecule associated with it including, for example, chromagenic or fluorogenic molecules.
  • a suitable substrate for use in the present invention is any substrate that produces an optically detectable signal upon interaction (e.g., reaction) with a given enzyme having a desired activity, or a given clone encoding such enzyme.
  • a suitable substrate based on a desired enzyme activity, for example.
  • desired enzymes/enzymatic activities include those listed herein.
  • a desired enzyme activity may also comprise a group of enzymes in an enzymatic pathway for which there exists an optical signal substrate.
  • This is the set of carotenoid synthesis enzymes.
  • Substrates are known and/or are commercially available for glycosidases, proteases, phosphtases, and monoxygenases, among others.
  • proteases with detectable (e.g., optical) signal substrates are the serine proteases — trypsin and chymotrypsin.
  • glucosidases are mannosidase, amyloglucosidase, cellulase, neuraminidase, beta-galactosidase, beta-glucosidase, beta-glucouronidase and alpha- amylase.
  • the desired activity is in the same class as that of other biomolecules or enzymes having a number known substrates
  • the activity can be examined using a cocktail of the known substrates.
  • substrates are known for approximately 20 commercially available esterases and the combination of these known substrate can provide detectable, if not optimal, signal production.
  • the optical signal substrate can be a chromogenic substrate, a fluorogenic substrate, a bio-or chemi-luminescent substrate, or a fluorescence resonance energy transfer (FRET) substrate.
  • the detectable species can be one which results from cleavage of the substrate or a secondary molecule which is so affected by the cleavage or other substrate/biomolecule interaction as to undergo a detectable change. Innumerable examples of detectable assay formats are known from the diagnostic arts which use immmunoassay, chromogenic assay, and labeled probe methodologies.
  • the optical signal substrate can be a bio- or chemi- luminescent substrate.
  • Chemiluminescent substrates for several enzymes are available from Tropix (Bedford, MA).
  • the enzymes having known chemiluminescent substrates are alkaline phosphatase, beta-galactosidase, beta-glucouronidase, and beta- glucosidase.
  • chromogenic substrates may be used, particularly for certain enzymes such as hydrolytic enzymes.
  • the optical signal substrate can be an indolyl derivative, which is enyzmatically cleaved to yield a chromogenic product.
  • the optically detectable signal is optical absorbance (including changes in absorbance).
  • signal detection can be provided by an absorbance measurement using a spectrophotometer or the like.
  • a fluorogenic substrate is used, such that the optically detectable signal is fluorescence.
  • Fluorogenic substrates provide high sensitivity for improved detection, as well as alternate detection modes. Hydroxy- and amino- substituted coumarins are the most widely used fluorophores used for preparing fluorogenic substrates.
  • a typical coumarin-based fluorogenic substrate is 7- hydroxycoumarin, commonly known as umbelliferone (Umb). Derivatives and analogs of umbelliferone are also used.
  • Substrate based on derivative and analogs of florescein (such as FDG or C12-FDG) and rhodamine are also used.
  • Substrates derived from resorufin are particularly useful in the present invention.
  • Resorufin-based substrates are useful, for example, in screening for glycosidases, hydrolases and dealkylases.
  • Lipophilic derivatives of the foregoing substrates e.g., alkylated derivatives
  • Fluorescein and resorufin are available commercially as alkylated derivatives that form products that are relatively insoluble in water (i.e., lipophilic).
  • fluorescence imaging can be performed using C12- resorufin galactoside, produced by Molecular Probes (Eugene, OR) as a substrate.
  • the particular fluorogenic substrate used may be chosen based on the enzymatic activity being screened. For examples:
  • Lipases/ esterases When screening for an enzyme having lipase or esterase activity, an acylated derivatives of fluorescein in used. The fluorophore is hydrolyzed from the derivative to generate a signal.
  • Enzymes having protease activity can be screened in the same way as the esterases, with an amide bond cleaved instead of an ester. There are now well over 100 different protease substrates available with an acylated fluorophore at the scissile bond. Rhodamine derivatives are generally used.
  • Monooxygenases (dealkylases).
  • Several coumarin derivatives suitable as monooxygenase substrates are commercially available. Typically, in these substrates, the hydroxylation of the ethyl group in the compound results in the release of the resorufin fluorophore.
  • the substrates are able to enter the cell and maintain its presence within the cell for a period sufficient for analysis to occur (e.g. , once the substrate is in the cell it does not "leak" back out before reacting with the enzyme being screened to an extend sufficient to produce a detectable response). Retention of the substrate in the cell canbe enhancedby a variety of techniques.
  • the substrate compound is structurally modified by addition of a hydrophobic (e.g., alkyl) tail.
  • a solvent such as DMSO or glycerol, can be used to coat the exterior of the cell.
  • the substrate can be administered to the cells at reduced temperature, which has been observed to retard leakage of substrates from cells.
  • the optical signal substrate can, in some embodiments, be a FRET substrate.
  • FRET is a spectroscopic method that can monitor proximity and relative angular orientation of fluorophores.
  • a fluorescent indicator system that uses FRET to measure the concentration of a substrate or products includes two fluorescent moieties having emission and excitation spectra that render one a "donor' fluorescent moiety and the other an "acceptor" fluorescent moiety.
  • the two fluorescent moieties are chosen such that the excitation spectrum of the acceptor fluorescent moiety overlaps with the emission spectrum of the excited moiety (the donor fluorescence moiety).
  • the donor moiety is excited by light of appropriate intensity within the excitation spectrum of the donor moiety and emits the absorbed energy as fluorescent light.
  • the acceptor fluorescent protein moiety is positioned to quench the donor moiety in the excited state, the fluorescence energy is transferred to the acceptor moiety, which can emit a second photon.
  • the emission spectra of the donor and acceptor moieties have minimal overlap so that the two emissions can be distinguished. Thus, when acceptor emits fluorescence at longer wavelength that the donor, then the net steady state effect is that the donor's emission is quenched , and the acceptor now emits when excited at the donor's absorption maximum.
  • the detectable or optical signal can be measured using, for example, a fluoremeter (or the like) to detect fluorescence, including fluorescence polarization, time-resolved fluorescence or FRET.
  • excitation radiation from an excitation source having a first wavelength, causes the excitation radiation to excite the sample.
  • fluorescence compounds in the sample emit radiation having a wavelength that is different from the excitation wavelength.
  • FDG fluorescein- di-beta-D-glactopyranoside
  • beta-galactosidase a substrate for beta-galactosidase
  • fluorescein a product that can be detected by its fluorescence emission upon hydrolysis
  • C12-FDG 5- dodecanoylamino fluorescein di-beta-Dgalactopyranside
  • Molecular Probes which differs from FDG in that it is a lipophilic fluorescein derivative that can easily cross most cell membranes under physiological culture conditions.
  • beta-galactosidase assays may be employed to screen single E. coli cells, expressing recombinant beta-D-galactosidase isolated, for example, from a hyperthermophilic archaeon such as Sulfolobus solfataricus.
  • Other reporter genes may be useful as substrates and are known for beta-glucouronidase, alkaline phosphatase, chloramphenical acetyltransferase (CAT) and luciferase.
  • the library may, for example, be screened for a specified enzyme activity.
  • the enzyme activity screened for may be one or more of the six IUB classes; oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
  • the recombinant enzymes which are determined to be positive for one or more of the IUB classes may then be rescreened for a more specific enzyme activity.
  • the library may be screened for a more specialized enzyme activity.
  • the library may be screened for a more specialized activity, i.e. the type of bond on which the hydrolase acts.
  • the library may be screened to ascertain those hydrolases which act on one or more specified chemical functionalities, such as: (a) amide (peptide bonds), i.e. proteases; (b) ester bonds, i.e. esterases and lipases; (c) acetals, i.e., glycosidases and the like.
  • the invention provides a process for activity screening of clones containing selected DNA derived from a microorganism which method includes:
  • the library includes a plurality of clones, the clones having been prepared by recovering nucleic acids (e.g., genomic DNA) from a mixed population of organisms, enriched populations thereof, or isolates thereof, and transforming a host with the nucleic acids to produce clones which are screened for the biomolecule or bioactivity of interest.
  • nucleic acids e.g., genomic DNA
  • an enrichment step may be used before activity based screening.
  • the enrichment step can be, for example, a biopanning method. This procedure of "biopanning" is described and exemplified in U.S. Patent No. 6,054,002, issued April 25, 2000, which is incorporated herein by reference.
  • polynucleotides are contained in clones, the clones having been prepared from nucleic acid sequences of a mixed population of organisms, wherein the nucleic acid sequences are used to prepare a gene library of the mixed population of organisms.
  • the gene library is screened for a sequence of interest by transfecting a host cell containing the library with at least one nucleic acid sequence having a detectable molecule which is all or a portion of a DNA sequence encoding a bioactivity having a desirable activity and separating the library clones containing the desirable sequence by, for example, a fluorescent based analysis.
  • the present invention offers the ability to screen for many types of bioactivities. For instance, the ability to select and combine desired components from a library of polyketides and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing.
  • the method(s) of the present invention make it possible to and facilitate the cloning of novel polyketide synthases, and other relevant pathways or genes encoding commercially relevant secondary metabolites, since one can generate gene libraries with clones containing large inserts (especially when using vectors which can accept large inserts, such as the f-factor based vectors), which facilitates cloning of gene clusters.
  • the biopanning approach described above can be used to create libraries enriched with clones carrying sequences homologous to a given probe sequence.
  • libraries containing clones with inserts of up to 40 kbp can be enriched approximately 1,000 fold after each round of panning. This enables one to reduce the number of clones to be screened after 1 round of biopanning enrichment.
  • This approach can be applied to create libraries enriched for clones carrying sequence of interest related to a bioactivity of interest for example polyketide sequences.
  • Hybridization screening using high density filters or biopanning has proven an efficient approach to detect homologues of pathways containing conserved genes.
  • Another approach of the present invention is to screen in E. coli for the expression of small molecule ring structures or "backbones". Because the genes encoding these polycyclic structures can often be expressed in E. coli the small molecule backbone can be manufactured albeit in an inactive form. Bioactivity is conferred upon transferring the molecule or pathway to an appropriate host that expresses the requisite glycosylation and methylation genes that can modify or "decorate" the structure to its active form. Thus, inactive ring compounds, recombinantly expressed in E.
  • coli are detected to identify clones which are then shuttled to a metabolically rich host, such as Streptomyces, for subsequent production of the bioactive molecule.
  • a metabolically rich host such as Streptomyces
  • One approach to detect and enrich for clones carrying these structures is to use the capillary screening methods or FACS screening, a procedure described and exemplified in U.S. Ser. No. 08/876,276, filed Jun. 16, 1997.
  • Polycyclic ring compounds typically have characteristic fluorescent spectra when excited by ultraviolet light.
  • clones expressing these structures can be distinguished from background using a sufficiently sensitive detection method.
  • high throughput FACS screening can be utilized to screen for small molecule backbones in E. coli libraries.
  • Commercially available FACS machines are capable of screening up to 100,000 clones per second for UV active molecules. These clones can be sorted for further FACS screening or the resident plasmids can be extracted and shuttled to Streptomyces for activity screemng.
  • organic extracts from candidate clones can be tested for bioactivity by susceptibility screening against test organisms such as Staphylococcus aureus, E. coli, or Saccharomyces cervisiae.
  • FACS screening can be used in this approach by co-encapsulating clones with the test organism.
  • mixed extract screening An alternative to the above-mentioned screening methods provided by the present invention is an approach termed “mixed extract” screening.
  • the “mixed extract” screening approach takes advantage of the fact that the accessory genes needed to confer activity upon the polycyclic backbones are expressed in metabolically rich hosts, such as Streptomyces, and that the enzymes can be extracted and combined with the backbones extracted from E. coli clones to produce the bioactive compound in vitro.
  • Enzyme extract preparations from metabolically rich hosts, such as Streptomyces strains, at various growth stages are combined with pools of organic extracts from E. coli libraries and then evaluated for bioactivity.
  • Another approach to detect activity in the E. coli clones is to screen for genes that can convert bioactive compounds to different forms. For example, a recombinant enzyme was recently discovered that can convert the low value daunomyciri to the higher value doxorubicin. Similar enzyme pathways are being sought to convert penicillins to cephalosporins.
  • Capillary screening can also be used to detect expression of UV fluorescent molecules in metabolically rich hosts, such as Streptomyces.
  • Recombinant oxytetracylin retains its diagnostic red fluorescence when produced heterologously in S. lividans TK24.
  • Pathway clones which can be identified by the methods and systems of the invention, can thus be screened for polycyclic molecules in a high throughput fashion.
  • Recombinant bioactive compounds can also be screened in vivo using "two- hybrid" systems, which can detect enhancers and inhibitors of protein-protein or other interactions such as those between transcription factors and their activators, or receptors and their cognate targets.
  • both a small molecule pathway and a GFP reporter construct are co-expressed. Clones altered in GFP expression can then be identified and the clone isolated for characterization.
  • the present invention also allows for the transfer of cloned pathways derived from uncultivated samples into metabolically rich hosts for heterologous expression and downstream screening for bioactive compounds of interest using a variety of screemng approaches briefly described above.
  • DNA can be isolated from positive clones utilizing techniques well known in the art.
  • the DNA can then be amplified either in vivo or in vitro by utilizing any of the various amplification techniques known in the art. In vivo amplification would include transformation of the clone(s) or subclone(s) into a viable host, followed by growth of the host. In vitro amplification can be performed using techniques such as the polymerase chain reaction. Once amplified the identified sequences can be "evolved" or sequenced.
  • One advantage afforded by present invention is the ability to manipulate the identified biomolecules or bioactivities to generate and select for encoded variants with altered sequence, activity or specificity.
  • Clones found to have biomolecules or bioactivities for which the screen was performed can be subjected to directed mutagenesis to develop new biomolecules or bioactivities with desired properties or to develop modified biomolecules or bioactivities with particularly desired properties that are absent or less pronounced in nature (e.g., wild-type activity), such as stability to heat or organic solvents.
  • Any of the known techniques for directed mutagenesis are applicable to the invention.
  • particularly preferred mutagenesis techniques for use in accordance with the invention include those described below.
  • a biomolecule e.g., a peptide, protein, or polynucleotide sequence
  • a bioactivity e.g., an enzymatic activity
  • Such variegation can modify the biomolecule or bioactivity in order to increase or decrease, for example, a polypeptide 's activity, specificity, affinity, function, and the like.
  • DNA shuffling can be used to increase variegation in a particular sample.
  • DNA shuffling is meant to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like (see, for example, U.S. Patent No. 5,939,250, issued to Dr. Jay Short on August 17, 1999, and assigned to Diversa Corporation, the disclosure of which is incorporated herein by reference).
  • Various methods for shuffling, mutating or variegating polynucleotide or polypeptide sequences are discussed below.
  • Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce a polynucleotide or polynucleotides. Mixtures of related nucleic acid sequences or polynucleotides are subjected to sexual PCR to provide random polynucleotides, and reassembled to yield a library or mixed population of recombinant hybrid nucleic acid molecules or polynucleotides. In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other than primers).
  • the advantage of the mutagenic shuffling of the invention over error-prone PCR alone for repeated selection can best be explained as follows.
  • the initial library of selected or pooled sequences can consist of related sequences of diverse origin or can be derived by any type of mutagenesis (including shuffling) of a single gene.
  • a collection of selected sequences is obtained after the first round of activity selection.
  • Shuffling allows the free combinatorial association of all of the related sequences, for example.
  • This method differs from error-prone PCR, in that it is an inverse chain reaction.
  • error-prone PCR the number of polymerase start sites and the number of molecules grows exponentially. However, the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same.
  • nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time. For polynucleotides derived from whole plasmids the theoretical endpoint is a single, large concatemeric molecule.
  • Rare shufflants will contain a large number of the best molecules (e.g., highest activity or specificity) and these rare shufflants may be selected based on their superior activity or specificity.
  • a pool of 100 different polypeptide sequences can be permutated in up to 10 3 different ways. This large number of permutations cannot be represented in a single library of DNA sequences. Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired. Error-prone PCR, in contrast, keeps all the selected sequences in the same relative orientation, generating a much smaller mutant cloud.
  • the template polynucleotide which may be used in the methods of the invention may be DNA or RNA. It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.
  • the template polynucleotide is from 50 bp to 50 kb. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of the invention, and in fact have been successfully used.
  • the template polynucleotide may be obtained by amplification using the PCR reaction (US Patent Nos. 4,683,202 and 4,683,195) or other amplification or cloning methods.
  • the removal of free primers from the PCR products before subjecting them to pooling of the PCR products and sexual PCR may provide more efficient results. Failure to adequately remove the primers from the original pool before sexual PCR can lead to a low frequency of crossover clones.
  • the template polynucleotide often is double-stranded.
  • a double-stranded nucleic acid molecule is recommended to ensure that regions of the resulting single-stranded polynucleotides are complementary to each other and thus can hybridize to form a double-stranded molecule.
  • single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, at this step. It is also contemplated that two different but related polynucleotide templates can be mixed at this step.
  • the double-stranded polynucleotide template and any added double-or single-stranded polynucleotides are subjected to sexual PCR which includes slowing or halting to provide a mixture of from about 5 bp to 5 kb or more.
  • the size of the random polynucleotides is from about 10 bp to 1000 bp, more preferably the size of the polynucleotides is from about 20 bp to 500 bp.
  • double-stranded nucleic acid having multiple nicks may be used in the methods of the invention.
  • a nick is a break in one strand of the double-stranded nucleic acid.
  • the distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp. This can provide areas of self-priming to produce shorter or smaller polynucleotides to be included with the polynucleotides resulting from random primers, for example.
  • the concentration of any one specific polynucleotide will not be greater than 1% by weight of the total polynucleotides, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.
  • the number of different specific polynucleotides in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.
  • single-stranded or double-stranded polynucleotides may be added to the random double-stranded shorter or smaller polynucleotides in order to increase the heterogeneity of the mixture of polynucleotides.
  • populations of double-stranded randomly broken polynucleotides may be mixed or combined at this step with the polynucleotides from the sexual PCR process and optionally subjected to one or more additional sexual PCR cycles.
  • single-stranded or double-stranded polynucleotides having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded polynucleotides may be added in a 10 fold excess by weight as compared to the total nucleic acid.
  • populations of polynucleotides from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1:40.
  • a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for).
  • the ratio of randomly provided wild-type polynucleotides which may be added to the randomly provided sexual PCR cycle hybrid polynucleotides is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.
  • the mixed population of random polynucleotides are denatured to form single-stranded polynucleotides and then re-annealed. Only those single-stranded polynucleotides having regions of homology with other single-stranded polynucleotides will re-anneal.
  • the random polynucleotides may be denatured by heating.
  • One skilled in the art could determine the conditions necessary to completely denature the double- stranded nucleic acid.
  • the temperature is from 80 °C to 100 °C, more preferably the temperature is from 90 °C to 96 °C.
  • Other methods which may be used to denature the polynucleotides include pressure and pH.
  • the polynucleotides may be re-annealed by cooling.
  • the temperature is from 20 °C to 75 °C, more preferably the temperature is from 40 °C to 65 °C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult.
  • the degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded polynucleotides.
  • Renaturation can be accelerated by the addition of polyethylene glycol
  • PEG poly(ethylene glycol) or salt.
  • the salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mm.
  • the salt may be KC1 or NaCl.
  • the concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
  • the annealed polynucleotides are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP, DGTP and dTTP).
  • a nucleic acid polymerase i.e. dATP, dCTP, DGTP and dTTP.
  • the nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.
  • the approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers. If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65 °C. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30 °C. One skilled in the art could vary the temperature of annealing to increase the number of cross-overs achieved.
  • the polymerase may be added to the random polynucleotides prior to annealing, simultaneously with annealing or after annealing.
  • the cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid.
  • This cycle is repeated for a desired number of times.
  • the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.
  • the resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.
  • This larger polynucleotides may contain a number of copies of a polynucleotide having the same size as the template polynucleotide in tandem.
  • This concatemeric polynucleotide is then denatured into single copies of the template polynucleotide.
  • the result will be a population of polynucleotides of approximately the same size as the template polynucleotide.
  • the population will be a mixed population where single or double-stranded polynucleotides having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.
  • These polynucleotides are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.
  • the single polynucleotides may be obtained from the larger concatemeric polynucleotide by amplification of the single polynucleotide prior to cloning by a variety of methods including PCR (US Patent Nos. 4,683,195 and 4,683,202), rather than by digestion of the concatemer.
  • the vector used for cloning is not critical provided that it will accept a polynucleotide of the desired size. If expression of the particular polynucleotide is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the polynucleotide to allow expression of the polynucleotide in the host cell.
  • the resulting bacterial population will include a number of recombinant polynucleotides having random mutations. This mixed population may be tested to identify the desired recombinant polynucleotides. The method of selection will depend on the polynucleotide desired.
  • a polynucleotide identified by the methods of described herein, encodes a protein with a first binding affinity
  • subsequent mutated (e.g., shuffled) sequences having an increased binding efficiency to a ligand may be desired.
  • the proteins expressed by each of the portions of the polynucleotides in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e. panning, affinity chromatography).
  • the proteins expressed by each of the polynucleotides in the population or library may be tested for their ability to confer drug resistance to the host organism.
  • One skilled in the art given knowledge of the desired protein, could readily test the population to identify polynucleotides which confer the desired properties onto the protein.
  • a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee WI).
  • the recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule.
  • the phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell.
  • the leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle.
  • the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.
  • a number of cycles of nucleic acid shuffling may be conducted with polynucleotides from a sub-population of the first population, which sub-population contains DNA encoding the desired recombinant protein. In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.
  • a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type polynucleotides and a sub-population of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the sub-population.
  • nucleic acid in a purified form can be utilized as the starting nucleic acid.
  • the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded.
  • a DNA-RNA hybrid which contains one strand of each may be utilized.
  • the nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably the specific nucleic acid sequence is from 50 to 50,000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of the invention.
  • Any specific nucleic acid sequence can be used to produce the population of hybrids by the present process. It is only necessary that a small population of hybrid sequences of the specific nucleic acid sequence exist or be available for the present process.
  • a population of specific nucleic acid sequences having mutations may be created by a number of different methods. Mutations may be created by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence.
  • mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence. The polynucleotide sequence can also be altered by chemical mutagenesis.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid, other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil,
  • 2-aminopurine, or acridine are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence.
  • Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
  • Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.
  • plasmid polynucleotides so mutagenized are introduced into E. coli and propagated as a pool or library of hybrid plasmids.
  • a small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes). Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.
  • the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.
  • the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of the invention.
  • the templates of the invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).
  • viruses e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like
  • selected portions thereof e.g., coat protein, spike glycoprotein, capsid protein.
  • cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large polynucleotides.
  • a mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified.
  • Utility can be readily determined by screening expressed polypeptides.
  • the DNA shuffling method of the invention can be performed blindly on a pool of unknown sequences.
  • any sequence mixture can be incorporated at any specific position into another sequence mixture.
  • mixtures of synthetic oligonucleotides, PCR polynucleotides or even whole genes can be mixed into another sequence library at defined positions.
  • the insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template.
  • the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.
  • Shuffling requires the presence of homologous regions separating regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling.
  • the conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle which are well-known in the art. This shuffling can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.
  • the equivalents of some standard genetic matings may also be performed by shuffling in vitro.
  • a "molecular backcross" can be performed by repeatedly mixing the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.
  • this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (e.g., immunogenicity).
  • unselected characteristics e.g., immunogenicity
  • DNA amplification by PCR typically includes sequences of about 40 kb. Amplification of a whole genome such as that of E. coli (5, 000 kb) by PCR would require about 250 primers yielding 125 forty kb polynucleotides.
  • random production of polynucleotides of the genome with sexual PCR cycles, followed by gel purification of small polynucleotides will provide a multitude of possible primers. Use of this mix of random small polynucleotides as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatamer containing many copies of the genome.
  • a 100 fold amplification in the copy number and an average polynucleotide size of greater than 50 kb may be obtained when only random polynucleotides are used. It is thought that the larger concatamer is generated by overlap of many smaller polynucleotides. The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA. It is expected that this approach will be useful for the mapping of genomes.
  • the polynucleotide to be shuffled can be produced as random or non-random polynucleotides, at the discretion of the practitioner.
  • the invention provides a method of shuffling that is applicable to a wide range of polynucleotide sizes and types, including the step of generating polynucleotide monomers to be used as building blocks in the reassembly of a larger polynucleotide.
  • the building blocks can be fragments of genes or they can be comprised of entire genes or gene pathways, or any combination thereof.
  • a mixed population of a specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell.
  • the polynucleotides can be introduced into the host cells by a variety of different methods.
  • the host cells can be transformed with the smaller polynucleotides using methods known in the art, for example treatment with calcium chloride. If the polynucleotides are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences.
  • the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.
  • specific nucleic acid sequences will be present in vectors which are capable of stably replicating the sequence in the host cell.
  • the vectors will encode a marker gene such that host cells having the vector can be selected. This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.
  • the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence. Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the polynucleotides into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein. It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.
  • the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired. For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected. If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand. Alternatively, the protein can be expressed in another system to ensure proper processing.
  • the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above.
  • This cycle can be repeated a number of times as desired.
  • a backcross can be performed in the second or subsequent recombination cycle.
  • a molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type sequence, such that at least one wild-type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation. Recombination with the wild-type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.
  • a subset of specific nucleic acid sequences can be generated as smaller polynucleotides by slowing or halting their PCR amplification prior to introduction into the host cell.
  • the size of the polynucleotides must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences.
  • the size of the polynucleotides will range from 0.03 kb to 100 kb more preferably from 0. 2 kb to 10 kb.
  • nucleic acid sequences other than the sequences selected from the previous round may be utilized to generate PCR polynucleotides prior to introduction into the host cells.
  • the shorter polynucleotide sequences can be single-stranded or double-stranded.
  • the reaction conditions suitable for separating the strands of nucleic acid are well known in the art.
  • the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. coli.
  • the particular vector is not essential, so long as it is capable of autonomous replication in E. coli.
  • the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector. It is also preferred that the vector contain a gene encoding for a selectable marker.
  • the population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells.
  • the vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage.
  • the concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell. Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in the generation of hybrids (daughters) having a combination of mutations which differ from the original parent mutated sequences.
  • the host cells are then clonally replicated and selected for the marker gene present on the vector. Only those cells having a plasmid will grow under the selection. The host cells which contain a vector are then tested for the presence of favorable mutations.
  • the nucleic acid is isolated either already linked to the vector or separated from the vector. This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.
  • the parent mutated specific nucleic acid population either as polynucleotides or cloned into the same vector is introduced into the host cells already containing the daughter nucleic acids. Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above. This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained. It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the hybrids may come from the parental hybrids or any subsequent generation.
  • the invention provides a method of conducting a
  • neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties. Such mutations may however confer on the nucleic acid or peptide undesirable characteristics. Accordingly, it is desirable to eliminate such neutral mutations.
  • the method of the invention provide a means of doing so.
  • the nucleic acid after the hybrid nucleic acid, having the desired characteristics, is obtained by the methods of he embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated.
  • the nucleic acid or vector is then introduced into the host cell with a large excess of the wild-type nucleic acid.
  • the nucleic acid of the hybrid and the nucleic acid of the wild-type sequence are allowed to recombine.
  • the resulting recombinants are placed under the same selection as the hybrid nucleic acid. Only those recombinants which retained the desired characteristics will be selected. Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA. This cycle can be repeated a number of times until all of the silent mutations are eliminated.
  • the invention provides for a method for shuffling, assembling, reassembling, recombining, and/or concatenating at least two polynucleotides to form a progeny polynucleotide (e.g., a chimeric progeny polynucleotide that can be expressed to produce a polypeptide or a gene pathway).
  • a progeny polynucleotide e.g., a chimeric progeny polynucleotide that can be expressed to produce a polypeptide or a gene pathway.
  • a double stranded polynucleotide e.g.
  • two single stranded sequences hybridized to each other as hybridization partners is treated with an exonuclease to liberate nucleotides from one of the two strands, leaving the remaining strand free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner.
  • a double stranded polynucleotide end (that may be part of- or connected to - a polynucleotide or a non-polynucleotide sequence) is subjected to a source of exonuclease activity.
  • Enzyme with 3' exonuclease activity, an enzyme with 5' exonuclease activity, an enzyme with both 3' exonuclease activity and 5' exonuclease activity, and any combination thereof can be used in the invention.
  • An exonuclease can be used to liberate nucleotides from one or both ends of a linear double stranded polynucleotide, and from one to all ends of a branched polynucleotide having more than two ends.
  • a non-enzymatic step may be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks that is comprised of subjecting a working sample to denaturing (or "melting") conditions (for example, by changing temperature, pH, and /or salinity conditions) so as to melt a working set of double stranded polynucleotides into single polynucleotide strands.
  • denaturing or "melting”
  • the presence of the former hybridization partners in the reaction vessel does not preclude, and may sometimes even favor, re-annealment of a single stranded polynucleotide with its former partner, to recreate an original double stranded polynucleotide.
  • the invention further provides an exonuclease-based approach requiring no denaturation - rather, the avoidance of denaturing conditions and the maintenance of double stranded polynucleotide substrates in annealed (i.e.
  • non-denatured state are necessary conditions for the action of exonucleases (e.g., exonuclease III and red alpha gene product).
  • exonucleases e.g., exonuclease III and red alpha gene product.
  • generation of single stranded polynucleotide sequences capable of hybridizing to other single stranded polynucleotide sequences is the result of covalent cleavage - and hence sequence destruction - in one of the hybridization partners.
  • an exonuclease III enzyme may be used to enzymatically liberate 3' terminal nucleotides in one hybridization strand (to achieve covalent hydrolysis in that polynucleotide strand); and this favors hybridization of the remaining single strand to a new partner (since its former partner was subjected to covalent cleavage).
  • enzymes can be discovered, optimized (e.g., engineered by directed evolution), or both discovered and optimized specifically for the instantly disclosed approach that have more optimal rates and/or more highly specific activities &/or greater lack of unwanted activities.
  • the invention may encourage the discovery and/or development of such designer enzymes.
  • a double stranded polynucleotide end having a 3' overhang is not susceptible to the exonuclease action of exonuclease III.
  • exonuclease III may be rendered susceptible to the exonuclease action of exonuclease III by a variety of means; for example, it may be blunted by treatment with a polymerase, cleaved to provide a blunt end or a 5' overhang, joined (ligated or hybridized) to another double stranded polynucleotide to provide a blunt end or a 5 ' overhang, hybridized to a single stranded polynucleotide to provide a blunt end or a 5' overhang, or modified by any of a variety of means).
  • an exonuclease may be allowed to act on one or on both ends of a linear double stranded polynucleotide and proceed to completion, to near completion, or to partial completion.
  • the exonuclease action is allowed to go to completion, the result will be that the length of each 5' overhang will be extend far towards the middle region of the polynucleotide in the direction of what might be considered a "rendezvous point" (which may be somewhere near the polynucleotide midpoint).
  • the exonuclease-mediated approach is useful for shuffling, assembling and or reassembling, recombining, and concatenating polynucleotide building blocks.
  • the polynucleotide building blocks can be up to ten bases long or tens of bases long or hundreds of bases long or thousands of bases long or tens of thousands of bases long or hundreds of thousands of bases long or millions of bases long or even longer.
  • Substrates for an exonuclease may be generated by subjecting a double stranded polynucleotide to fragmentation. Fragmentation may be achieved by mechanical means (e.g., shearing, sonication, and the like), by enzymatic means (e.g., using restriction enzymes), and by any combination thereof. Fragments of a larger polynucleotide may also be generated by polymerase-mediated synthesis.
  • Red alpha (redd) gene product also referred to as lambda exonuclease
  • Red alpha gene product acts processively from 5'-phosphorylated termini to liberate mononucleotides from duplex DNA (Takahashi & Kobayashi, 1990).
  • Venom phosphodiesterases (Laskowski, 1980) is capable of rapidly opening supercoiled DNA.
  • the present invention provides a non-stochastic method .termed synthetic ligation reassembly (SLR), that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or chimerized randomly, but rather are assembled non-stochastically.
  • SLR synthetic ligation reassembly
  • the SLR method does not depend on the presence of a high level of homology between polynucleotides to be shuffled.
  • the invention can be used to non- stochastically generate libraries (or sets) of progeny molecules comprised of over 10 ° different chimeras. Conceivably, SLR can even be used to generate libraries comprised of over io 1000 different progeny chimeras.
  • the invention provides a non-stochastic method of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating, by design, a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
  • the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
  • the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and, if more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
  • the annealed building pieces are treated with an enzyme, such as a ligase (e.g., T4 DNA ligase) to achieve covalent bonding of the building pieces.
  • a ligase e.g., T4 DNA ligase
  • the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules.
  • progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, ie. chimerized or shuffled.
  • the invention provides for the chimerization of a family of related genes and their encoded family of related products.
  • the encoded products are enzymes.
  • the following enzymes and their functions Lipase/Esterase, Protease, Glycosidase/Glycosyl, transferase, Phosphatase/Kinase, Mono/Dioxygenase, Haloperoxidase, Lignin, peroxidase/Diarylpropane peroxidase, Epoxide hydrolase, Nitrile hydratase/nitrilase, Transaminase, Amidase/Acylase.
  • the sequences of a plurality of progenitor nucleic acid templates identified using the methods of the invention are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology.
  • the demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be generated.
  • the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the progeny molecules.
  • a demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two progenitor templates, but the demarcation point can be an area of homology that is shared by at least half of the progenitor templates, at least two thirds of the progenitor templates, at least three fourths of the progenitor templates, and preferably at almost all of the progenitor templates. Even more preferably still a demarcation point is an area of homology that is shared by all of the progenitor templates.
  • the ligation reassembly process is performed exhaustively in order to generate an exhaustive library.
  • the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
  • the assembly order is by design (or non-stochastic). Because of the non-stochastic nature of the invention, the possibility of unwanted side products is greatly reduced.
  • the invention provides that, the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened systematically, e.g., one by one.
  • the invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, an experimental design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups.
  • the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules.
  • the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
  • such a generated library is comprised of greater than 10 3 to greater than l ⁇ 1000 different progeny molecular species.
  • a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide.
  • this polynucleotide is a gene, which may be a man-made gene.
  • this polynucleotide is a gene pathway, which may be a man-made gene pathway.
  • the invention provides that one or more man-made genes generated by the invention may be incorporated into a man-made gene pathway, such as pathway operable in a eukaryotic organism (including a plant).
  • the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g., by mutagenesis) or in an in vivo process (e.g., by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a demarcation point.
  • nucleotides e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences
  • the invention provides that a nucleic acid building block can be used to introduce an intron.
  • the invention provides that functional introns may be introduced into a man-made gene of the invention.
  • the invention also provides that functional introns may be introduced into a man-made gene pathway of the invention.
  • the invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced intron(s).
  • the invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced intron(s).
  • the artificially introduced intron(s) are functional in one or more host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.
  • the invention provides a process of producing man-made infron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing.
  • a man-made gene produced using the invention can also serve as a substrate for recombination with another nucleic acid.
  • a man-made gene pathway produced using the invention can also serve as a substrate for recombination with another nucleic acid.
  • the recombination is facilitated by, or occurs at, areas of homology between the man-made infron-containing gene and a nucleic acid with serves as a recombination partner.
  • the recombination partner may also be a nucleic acid generated by the invention, including a man-made gene or a man-made gene pathway. Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced intron(s) in the man-made gene.
  • the synthetic ligation reassembly method of the invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends.
  • the two ligatable ends on each nucleic acid building block may be two blunt ends (i.e. each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.
  • An overhang for this purpose may be a 3' overhang or a 5' overhang.
  • a nucleic acid building block may have a 3' overhang or alternatively a 5' overhang or alternatively two 3' overhangs or alternatively two 5' overhangs.
  • the overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by purposeful experimental design and is not random.
  • a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single-stranded oligos) and contacting them so as to allow them to anneal to form a double-stranded nucleic acid building block.
  • a double-stranded nucleic acid building block can be of variable size.
  • the sizes of these building blocks can be small or large.
  • Preferred sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs).
  • Other preferred size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100, 000 bp (including every integer value in between).
  • Figure 6 shows the activity of the enzyme exonuclease III.
  • This is an exemplary enzyme that can be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks. The asterisk indicates that the enzyme acts from the 3' direction towards the 5' direction of the polynucleotide substrate.
  • Figure 7 shows an exemplary application of the enzyme exonuclease III in exonuclease-mediated polynucleotide reassembly. Shown also is the combined use of in vivo "repair" by transforming a suitable host (e.g.
  • Figure 8 shows the generation of a poly-binding nucleic acid strand.
  • the generated strand is of the same length as the parental template, but is not methylated. Dpn I treatment can thus be used to select for the generated strand.
  • the template as well as the generated product can be part of a vector (linear or circular).
  • Figure 9 shows the use of exonuclease treatment as a means to liberate 3' and 5 '-terminal nucleotides from the unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but hybridized end to facilitate polymerase-based extension and/or ligase- mediated ligation of the treated end.
  • Figure 10 shows the use of exonuclease-mediated nucleic acid reassembly in an example in which one methylated poly-binding nucleic acid strand is annealed to several unmethylated mono-binding nucleic acid strands.
  • the annealed nucleic acid strands form heteromeric nucleic acid complexes and are subjected to exonuclease treatment as a means to liberate 3' and 5 '-terminal nucleotides from the unhybridized single-stranded ends of a plurality of annealed nucleic acid strands in the heteromeric nucleic acid complexes, leaving shortened but hybridized ends to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated ends.
  • Treatment with Dpnl is then serviceable for selecting against the generated annealed mono-binding nucleic acid strands that are unmethylated and chimeric in nature.
  • Figure 11 shows the use of exonuclease-mediated nucleic acid reassembly in an example in which a plurality of methylated poly-binding nucleic acid strand are annealed to several unmethylated mono-binding nucleic acid strands.
  • the annealed nucleic acid strands form heteromeric nucleic acid complexes that are subjected to exonuclease treatment as a means to liberate 3' and 5 '-terminal nucleotides from the unhybridized single-stranded ends of a plurality of annealed nucleic acid strands in the heteromeric nucleic acid complexes, leaving shortened but hybridized ends to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated ends.
  • Treatment with Dpnl is then serviceable for selecting against the generated annealed mono-binding nucleic acid strands that are unmethylated and chimeric in nature.
  • a double-stranded nucleic acid building block is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block.
  • the two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang; thus containing no mismatches, apart from any overhang(s).
  • the two strands of a double-stranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang.
  • a double-stranded nucleic acid building block can be used to introduce codon degeneracy.
  • the codon degeneracy is introduced using the site- saturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes.
  • Figure 1 shows a preferred embodiment of site-saturated mutagenesis as described herein.
  • Nucleic acid sequences that can be mutagenized by non-stochastic site-saturation mutagenesis include any polynucleotide of 15-100,000 bases in length (e.g. any coding region and any non-coding region).
  • such nucleic acid sequences include promoters, enhancers, introns, whole genes, cDNA, gene segments, operators, polynucleotide functional groups, expression regulatory sequences, expressed sequence tags (ESTs), open reading frames (ORFs), rpressors/transactivators, origin of replication, or gene pathways by way of example.
  • ESTs expressed sequence tags
  • ORFs open reading frames
  • rpressors/transactivators origin of replication, or gene pathways by way of example.
  • Mutagenic cassettes that can be mutagenized by non-stochastic site-saturation mutagenesis include any polynucleotide cassette of 1-500 bases in length.
  • Site- saturation mutagenesis is servicable for mutagenizing a complete set of cassettes contained within a polynucleotide sequence to be mutagenized.
  • cassettes can be spaced along each polynucleotide differently (i.e. immediately adjacent, evenly spaced, or unevenly spaced) and of any size.
  • a set of mutagenic cassettes is a set of contiguous codons within a sequence of defined length.
  • a set of mutagenic cassettes is a set of nucleotide cassettes that are not shared by aligned related polynucleotides.
  • the type of mutations to be introduced in a set of mutagenic cassettes can be of the same type or of different types within each round of polynucleotide site- saturation mutagenesis.
  • Each mutagenic cassette (within the nucleic acid sequence to be mutagenized) preferably is usually mutagenized by the use of a corresponding oligo (including by a degenerate oligo).
  • degenerate mutations provided by this invention include: Codons for all 20 amino acids (e.g. N 5 N,N or N,N,G/T or N,N,G/C) All degenerate codons that do not change the amino acid sequence of the parental template (i.e. codons for the same amino acid that is present in the parental template) Codons (all or selected) for amino acids within the same grouping according to the selected amino acid grouping scheme*.
  • Codons for at least 1 amino acid in each amino acids group* are Codons for at least 1 amino acid in each amino acids group*.
  • Acidic (Asp, Glu, Asn, Gin)
  • Figure 2 showcases the power of this invention in its ability to generate exhaustively and systematically all possible combinations of the nucleic acid building blocks designed in this example. Particularly large sets (or libraries) of progeny chimeric molecules can be generated. Because this method can be performed exhaustively and systematically, the method application can be repeated by choosing new demarcation points and with correspondingly newly designed nucleic acid building blocks, bypassing the burden of re-generating and re-screening previously examined and rejected molecular species. It is appreciated that, codon wobble can be used to advantage to increase the frequency of a demarcation point.
  • a particular base can often be substituted into a nucleic acid building block without altering the amino acid encoded by progenitor codon (that is now altered codon) because of codon degeneracy.
  • demarcation points are chosen upon alignment of 8 progenitor templates.
  • Nucleic acid building blocks including their overhangs are then designed and synthesized.
  • 18 nucleic acid building blocks are generated based on the sequence of each of the 8 progenitor templates, for a total of 144 nucleic acid building blocks (or double-stranded oligos). Performing the ligation reassembly procedure will then produce a library of progeny molecules comprised of yield of 818 (or over 1.8 x 1016) chimeras.
  • the in vivo recombination method of the invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence. However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide.
  • the approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA and growth hormone.
  • This approach may be used to generate v proteins having altered specificity or activity.
  • the approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes.
  • This approach may be used to generate genes having increased rates of expression.
  • This approach may also be useful in the study of repetitive DNA sequences.
  • this approach may be useful to mutate ribozymes or aptamers.
  • the invention provides a method for selecting a subset of polynucleotides from a starting set of polynucleotides, which method is based on the ability to discriminate one or more selectable features (or selection markers) present anywhere in a working polynucleotide, so as to allow one to perform selection for (positive selection) and/or against (negative selection) each selectable polynucleotide.
  • a method is provided termed end-selection, which method is based on the use of a selection marker located in part or entirely in a terminal region of a selectable polynucleotide, and such a selection marker may be termed an "end-selection marker".
  • End-selection may be based on detection of naturally occurring sequences or on detection of sequences introduced experimentally (including by any mutagenesis procedure mentioned herein and not mentioned herein) or on both, even within the same polynucleotide.
  • An end-selection marker can be a structural selection marker or a functional selection marker or both a structural and a functional selection marker.
  • An end-selection marker may be comprised of a polynucleotide sequence or of a polypeptide sequence or of any chemical structure or of any biological or biochemical tag, including markers that can be selected using methods based on the detection of radioactivity, of enzymatic activity, of fluorescence, of any optical feature, of a magnetic property (e.g., using magnetic beads), of immunoreactivity, and of hybridization.
  • End-selection may be applied in combination with any method for performing mutagenesis.
  • mutagenesis methods include, but are not limited to, methods described herein (supra and infra). Such methods include, by way of non-limiting exemplification, any method that may be referred herein or by others in the art by any of the following terms: “saturation mutagenesis”, “shuffling”, “recombination”, “re- assembly”, “error-prone PCR”, “assembly PCR”, “sexual PCR”, “crossover PCR”, “oligonucleotide primer-directed mutagenesis”, “recursive (and/or exponential) ensemble mutagenesis (see Arkin and Youvan, 1992)", “cassette mutagenesis”, “in vivo mutagenesis”, and “in vitro mutagenesis”.
  • end-selection may be performed on molecules produced by any mutagenesis and/or amplification method (see, e.g., Arnold, 1993; Caldwell and Joyce, 1992; Stemmer, 1994) following which method it is desirable to select for (including to screen for the presence of) desirable progeny molecules.
  • end-selection may be applied to a polynucleotide apart from any mutagenesis method.
  • end-selection as provided herein, can be used in order to facilitate a cloning step, such as a step of ligation to another polynucleotide (including ligation to a vector).
  • the invention thus provides for end- selection as a means to facilitate library construction, selection and/or enrichment for desirable polynucleotides, and cloning in general.
  • end-selection can be based on (positive) selection for a polynucleotide; alternatively end-selection can be based on (negative) selection against a polynucleotide; and alternatively still, end-selection can be based on both (positive) selection for, and on (negative) selection against, a polynucleotide.
  • End- selection, along with other methods of selection and/or screening, can be performed in an iterative fashion, with any combination of like or unlike selection and/or screening methods and mutagenesis or directed evolution methods, all of which can be performed in an iterative fashion and in any order, combination, and permutation.
  • end-selection may also be used to select a polynucleotide in a: circular (e.g., a plasmid or any other circular vector or any other polynucleotide that is partly circular), and/or branched, and/or modified or substituted with any chemical group or moiety.
  • a: circular e.g., a plasmid or any other circular vector or any other polynucleotide that is partly circular
  • branched e.g., a plasmid or any other circular vector or any other polynucleotide that is partly circular
  • end-selection of a linear polynucleotide is performed using a general approach based on the presence of at least one end- selection marker located at or near a polynucleotide end or terminus (that can be either a 5 ' end or a 3 ' end).
  • end- selection is based on selection for a specific sequence at or near a terminus such as, but not limited to, a sequence recognized by an enzyme that recognizes a polynucleotide sequence.
  • An enzyme that recognizes and catalyzes a chemical modification of a polynucleotide is referred to herein as a polynucleotide-acting enzyme.
  • polynucleotide-acting enzymes are exemplified non-exclusively by enzymes with polynucleotide-cleaving activity, enzymes with polynucleotide-methylating activity, enzymes with polynucleotide-ligating activity, and enzymes with a plurality of distinguishable enzymatic activities (including non- exclusively, e.g., both polynucleotide-cleaving activity and polynucleotide-ligating activity).
  • relevant polynucleotide-acting enzymes include any enzymes identifiable by one skilled in the art (e.g. , commercially available) or that may be developed in the future, though currently unavailable, that are useful for generating a ligation compatible end, preferably a sticky end, in a polynucleotide. It may be preferable to use restriction sites that are not contained, or alternatively that are not expected to be contained, or alternatively that are unlikely to be contained (e.g., when sequence information regarding a working polynucleotide is incomplete) internally in a polynucleotide to be subjected to end-selection. It is recognized that methods (e.g., mutagenesis methods) can be used to remove unwanted internal restriction sites.
  • a partial digestion reaction i.e. a digestion reaction that proceeds to partial completion
  • a digestion reaction can be used to achieve digestion at a recognition site in a terminal region while sparing a susceptible restriction site that occurs internally in a polynucleotide and that is recognized by the same enzyme.
  • partial digest are useful because it is appreciated that certain enzymes show preferential cleavage of the same recognition sequence depending on the location and environment in which the recognition sequence occurs.
  • protection methods can be used to selectively protect specified restriction sites (e.g., internal sites) against unwanted digestion by enzymes that would otherwise cut a working polypeptide in response to the presence of those sites; and that such protection methods include modifications such as methylations and base substitutions (e.g., U instead of T) that inhibit an unwanted enzyme activity.
  • specified restriction sites e.g., internal sites
  • modifications such as methylations and base substitutions (e.g., U instead of T) that inhibit an unwanted enzyme activity.
  • a useful end-selection marker is a terminal sequence that is recognized by a polynucleotide-acting enzyme that recognizes a specific polynucleotide sequence.
  • useful polynucleotide-acting enzymes also include other enzymes in addition to classic type II restriction enzymes.
  • useful polynucleotide-acting enzymes also include gyrases (e.g., topoisomerases), helicases, recombinases, relaxases, and any enzymes related thereto.
  • end-selection can be used to distinguish and separate parental template molecules (e.g., to be subjected to mutagenesis) from progeny molecules (e.g., generated by mutagenesis).
  • a first set of primers lacking in a topoisomerase I recognition site, can be used to modify the terminal regions of the parental molecules (e.g., in polymerase-based amplification).
  • a different second set of primers (e.g., having a topoisomerase I recognition site) can then be used to generate mutated progeny molecules (e.g., using any polynucleotide chimerization method, such as interrupted synthesis, template-switching polymerase-based amplification, or interrupted synthesis; or using saturation mutagenesis; or using any other method for introducing a topoisomerase I recognition site into a mutagenized progeny molecule) from the amplified template molecules.
  • the use of topoisomerase I-based end-selection can then facilitate, not only discernment, but selective topoisomerase I-based ligation of the desired progeny molecules.
  • Figure 3 is a diagram of the application of end-selection using a topoisomerase- guided selection step to select for desirable polynucleotides (e.g. the selection of full length molecules) generated by polynucleotide reassembly (e.g. by synthetic ligation reassembly).
  • Figure 4 illustrates various aspects of the end-selection technology.
  • Figure 5 illustrates a specific aspect of the end-selection technology where step of "c) Perfo ⁇ riing directed expression cloning" is achieved using the enzyme N.BstNBI in order to introduce "sticky ends" into an originally blunt-ended DNA in order to achieve directional expression cloning of the original blunt-ended DNA.
  • the present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype (e.g., for affinity for a predetermined receptor (ligand).
  • a phenotype e.g., for affinity for a predetermined receptor (ligand).
  • peptide structures including the primary amino acid sequences, of peptides or peptidomimetics that interact with biological macromolecules.
  • One method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor) involves the screening of a large library or peptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the peptide.
  • each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences.
  • Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.
  • a well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein.
  • the bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule.
  • the bacteriophage particles i.e., library members
  • the bacteriophage particles which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage sub-population for a subsequent round of affinity enrichment and phage replication.
  • the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor).
  • the predetermined macromolecule e.g., receptor
  • the present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion.
  • useful compounds e.g., peptides, including single-chain antibodies
  • the random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.
  • the mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or incorporation in a cell.
  • a significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest.
  • the peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.
  • the invention also provides a method for shuffling a pool of polynucleotide sequences identified by the methods of the invention and selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g. , a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).
  • a predetermined receptor e.g. , a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like
  • Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
  • the recombined selected polynucleotide sequences are subjected to at least one subsequent selection round.
  • the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection.
  • Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by back-crossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic. Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).
  • the sequences Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized.
  • selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced.
  • Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member.
  • sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence.
  • the mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.
  • the invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).
  • the invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g.
  • ligand or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members
  • shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library
  • the present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g. , for affinity for binding a predetermined antigen (ligand)).
  • a phenotype e.g. , for affinity for binding a predetermined antigen (ligand)
  • variable segment genes located upstream of a tandem array of diversity segment genes, which are themselves located upstream of a tandem array of joining (i) region genes, which are located upstream of the constant region genes.
  • V-D-J rearrangement occurs wherein a heavy chain variable region gene (VH) is formed by rearrangement to form a fused D segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (VH) of a heavy chain.
  • VH heavy chain variable region gene
  • VL variable region
  • variable regions possible in immuno globulins derives in part from the numerous combinatorial possibilities of joining V and i segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development. Additional sequence diversity in the heavy chain variable regions arises from non-uniform rearrangements of the D segments during V-D-J joining and from N region addition. Further, antigen-selection of specific B cell clones selects for higher affinity variants having non-germline mutations in one or both of the heavy and light chain variable regions; a phenomenon referred to as "affinity maturation" or "affinity sharpening".
  • CDRs complementarity-determining regions
  • Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al, 1989); Caton and Koprowski, 1990; Mullinax et al, 1990; Persson et al, 1991).
  • bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang et al, 1991; Clackson et al, 1991; McCafferty et al, 1990; Burton et al, 1991; Hoogenboom et al, 1991; Chang et al, 1991; Breifiing et al, 1991; Marks et al, 1991, p.
  • a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).
  • a receptor e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid
  • immobilized e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography
  • labeled e.g., to screen plaque or colony lifts
  • the first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.
  • the single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3)) or equivalent linker peptide(s).
  • the linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH'
  • the scfv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.
  • scfv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide.
  • the scfv genes are assembled, they are cloned into a phagemid and expressed at the tip of the Ml 3 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage PHI (gene 3) coat protein. Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scfv for binding to a predetermined epitope (e.g., target antigen, receptor).
  • a predetermined epitope e.g., target antigen, receptor
  • the linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence.
  • the displayed peptide (s) or single-chain antibody (e.g., scfv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system.
  • polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, ⁇ and the like.
  • polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like.
  • Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.
  • neoplasia a disease that causes inflammation
  • neoplasia a malignant neoplasia
  • autoimmune disease a malignant neoplasia
  • AIDS a malignant neoplasia
  • cardiovascular disease a malignant neoplasia .
  • infections a malignant neoplasia .
  • stage disease e.g., neoplasia
  • therapeutic application to treat disease such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.
  • VH and VL cassettes can themselves be diversified, such as by random, pseudorandom, or directed mutagenesis.
  • VH and VL cassettes are diversified in or near the complementarity-determining regions (CDRS), often the third CDR, CDR3.
  • Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scfv site-directed hybrids (Stemmer et al, 1993), as has error-prone PCR and chemical mutagenesis (Deng et al, 1994).
  • Riechmann Riechmann et al, 1993
  • Barbas (Barbas et al, 1992) attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.
  • CDR randomization has the potential to create approximately 1 x 10 20 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants.
  • the combination possibilities of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding. Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems.
  • Barbas Barbas et al, 1992
  • 5 x 10 7 transformants which represents only a tiny fraction of the potential diversity of a library of thoroughly randomized CDRs.
  • bacteriophage. display of scfv have already yielded a variety of useful antibodies and antibody fusion proteins.
  • a bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al, 1994).
  • Intracellular expression of an anti-Rev scfv has been shown to inhibit HIV-1 virus replication in vitro (Duan et al, 1994), and intracellular expression of an anti-p21rar, scfv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al, 1993).
  • scfv which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility of scfv (Lilley et al, 1994). Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al, 1992; Nicholls et al, 1993).
  • the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies.
  • Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s).
  • the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/12227, and WO 94/25585), including hybridomas derived therefrom.
  • antibody-producing cells e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/12227, and WO 94/25585
  • Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
  • the recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round.
  • the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained.
  • Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by back-crossing with a human variable region framework to produce human-like sequence antibodies.
  • neutral antibody framework sequences i.e., having insubstantial functional effect on antigen binding
  • back-crossing with a human variable region framework to produce human-like sequence antibodies.
  • subsequent selection is applied to retain the property of binding to the predetermined antigen.
  • the valency of the target epitope may be varied to control the average binding affinity of selected scfv library members.
  • the target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art.
  • a high density (valency) of predetermined epitope can be used to enrich for scfv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scfv library members.
  • a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kernal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR).
  • a predetermined site e.g., a CDR
  • the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like.
  • the resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i e. , may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.
  • Displayed peptide/polynucleotide complexes which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique. This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species. Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.
  • the library members without the desired specificity are removed by washing.
  • the degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope.
  • a certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing.
  • the temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule. Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route.
  • nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.
  • affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.
  • One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scfv library can be simultaneously screened for a multiplicity of scfv which have different binding specificities.
  • multiple binding targets multiple epitope species, multiple receptor species
  • a scfv library can be simultaneously screened for a multiplicity of scfv which have different binding specificities.
  • multiple target epitope species each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scfv library under suitable binding conditions.
  • the collection of beads, comprising multiple epitope species can then be used to isolate, by affinity selection, scfv library members.
  • subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species. This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.
  • a variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope.
  • the positive selected peptide/polynucleotide complexes are sequenced to determine the identity of the active peptides.
  • Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences.
  • This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations.
  • This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled. It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.
  • mutagenic PCR amplification of positive selected peptide/polynucleotide complexes is done prior to sequencing.
  • the same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling.
  • shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included.
  • mixtures of synthetic oligonucleotides and PCR produced polynucleotides can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).
  • the invention of shuffling enables the generation of a vast library of
  • CDR-variant single-chain antibodies One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling.
  • the sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences.
  • a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat (Kabat et al, 1991); the pool (s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence.
  • a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are incorporated into the synthetic CDRs at the corresponding position(s).
  • oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized.
  • a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids.
  • residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences.
  • Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences. Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.
  • the collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence. It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions.
  • a collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1 x 10 6 unique CDR sequences are included in the collection, although occasionally 1 x 10 to 1 x 10 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare (i.e., less than 0.1 percent) in that position in naturally-occurring human CDRS.
  • the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of 10.
  • Such single-chain antibodies generally bind of about at least 1 x 10 m-, preferably with an affinity of about at least 5 x 10 7 M-l, more preferably with an affinity of at least 1 x 10 8 M-l to 1 x 10 9 M-l or more, sometimes up to 1 x 10 10 M-l or more.
  • the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, L-selectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.
  • a human cell surface antigen e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor
  • other human biological macromolecule e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, L-selectin
  • nonhuman disease associated macromolecule e.g., bacterial LPS, virion capsid protein or envelope glycoprotein
  • High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems.
  • scfv have been produced in plants (Firek et al, 1993) and can be readily made in prokaryotic systems (Owens and Young, 1994; Johnson and Bird, 1991).
  • the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al, 1994).
  • the variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized.
  • the polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.
  • a host cell e.g., from an expression vector in a mammalian cell
  • the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, 1982).
  • polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Lefkovits and Pernis, 1979 and 1981; Lefkovits, 1997).
  • the antibodies generated by the method of the present invention can be used for diagnosis and therapy.
  • they can be used to treat cancer, autoimmune diseases, or viral infections.
  • the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.
  • Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence.
  • the selected library members are pooled and shuffled by in vitro and/or in vivo recombination.
  • the shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetermined polypeptide sequence (e.g., an SH2 domain from another protein species).
  • Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell. Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacz, HIS3) which is operably linked to a Gal4 binding site.
  • a reporter gene e.g., lacz, HIS3
  • the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver and Hunt, 1993; Durfee et al, 1993; Yang et al, 1992; Luban et al, 1993; Hardy et al, 1992; Bartel et al, 1993; and Vojtek et al, 1993).
  • variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li and Fields, 1993; Lalo et al, 1993; Jackson et al, 1993; and Madura et al, 1993).
  • Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al, 1993; Chakrabarty et al, 1992; Staudinger et al, 1993; and Milne and Weaver 1993) or domains responsible for oligomerization of a single protein (Iwabuchi et al, 1993; Bogerd et al, 1993). Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al, 1992). Alternatively, an E.
  • coli/ C P interactive screening system (Germino et al, 1993; Guarente, 1993) can be used to identify interacting protein sequences (i.e., protein sequences which heterodimerize or form higher order heteromultimers). Sequences selected by a two- hybrid system can be pooled and shuffled and introduced into a two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence. The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kernals.
  • One microgram samples of template DNA are obtained and treated with UN. light to cause the formation of dimers, including TT dimers, particularly purine dimers.
  • U.V. exposure is limited so that only a few photoproducts are generated per gene on the template DNA sample.
  • Multiple samples are treated with UN. light for varying periods of time to obtain template D ⁇ A samples with varying numbers of dimers from UN. exposure.
  • a random priming kit which utilizes a non-proofreading polymease for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems
  • a non-proofreading polymease for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems
  • the priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers.
  • the dimers formed by UN. exposure serve as a roadblock for the extension by the non-proofreading polymerase.
  • a pool of random size polynucleotides is present after extension with the random primers is finished.
  • the invention is further directed to a method for generating a selected mutant polynucleotide sequence (or a population of selected polynucleotide sequences) typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequences(s) possess at least one desired phenotypic characteristic (e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like) which can be selected for.
  • a desired phenotypic characteristic e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like
  • One method for identifying hybrid polypeptides that possess a desired structure or functional property involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.
  • a predetermined biological macromolecule e.g., a receptor
  • the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.
  • the method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequences, optimally introducing mutations into said polynucleotides or copies, (2) pooling the polynucleotides or copies, (3) producing smaller or shorter polynucleotides by interrupting a random or particularized priming and synthesis process or an amplification process, and (4) performing amplification, preferably PCR amplification, and optionally mutagenesis to homologously recombine the newly synthesized polynucleotides.
  • the means for blocking or interrupting the amplification or synthesis process is by utilization of UV light, DNA adducts, DNA binding proteins.
  • the DNA adducts, or polynucleotides comprising the DNA adducts are removed from the polynucleotides or polynucleotide pool, such as by a process including heating the solution comprising the DNA fragments prior to further processing.
  • clones which are identified as having a biomolecule or bioactivity of interest may also be sequenced to identify the DNA sequence encoding a polypeptide (e.g., an enzyme) or the polypeptide sequence itself having the specified activity, for example.
  • a polypeptide e.g., an enzyme
  • the polypeptide sequence itself having the specified activity for example.
  • Suitable clones (e.g., 1-1000 or more clones) from the library are identified by the methods of the invention and sequenced using, for example, high through-put sequencing techniques.
  • the exact method of sequencing is not a limiting factor of the invention. Any method useful in identifying the sequence of a particular cloned DNA sequence can be used.
  • sequencing is an adaptation of the natural process of DNA replication. Therefore, a template (e.g., the vector) and primer sequences are used.
  • One general template preparation and sequencing protocol begins with automated picking of bacterial colonies, each of which contains a separate DNA clone which will function as a template for the sequencing reaction. The selected clones are placed into media, and grown overnight. The DNA templates are then purified from the cells and suspended in water. After DNA quantification, high-throughput sequencing is performed using a sequencers, such as Applied Biosystems, Inc., Prism 377 DNA Sequencers. The resulting sequence data can then be used in additional methods, including searching a database or databases.
  • a number of source databases are available that contain either a nucleic acid sequence and/or a deduced amino acid sequence for use with the invention in identifying or determining the activity encoded by a particular polynucleotide sequence. All or a representative portion of the sequences (e.g., about 100 individual clones) to be tested are used to search a sequence database (e.g., GenBank, PFAM or ProDom), either simultaneously or individually. A number of different methods of performing such sequence searches are known in the art.
  • the databases can be specific for a particular organism or a collection of organisms. For example, there are databases for the C. elegans, Arabadopsis. sp., M. genitalium, M.jannaschii, E. coli, H. influenzae, S. cerevisiae and others.
  • the sequence data of the clone is then aligned to the sequences in the database or databases using algorithms designed to measure homology between two or more sequences.
  • sequence alignment methods include, for example, BLAST (Altschul et al, 1990), BLITZ (MPsrch) (Srurrock & Collins, 1993), and FASTA (Person & Lipman, 1988).
  • the probe sequence e.g., the sequence data from the clone
  • the threshold value may be predetermined, although this is not required.
  • the threshold value can be based upon the particular polynucleotide length.
  • To align sequences a number of different procedures can be used. Typically, Smith- Waterman or Needleman- Wunsch algorithms are used. However, as discussed faster procedures such as BLAST, FASTA, PSI-BLAST can be used.
  • optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI, or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, WI), or by inspection, and the best alignment (i. e. , resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • the similarity of the two sequence i.e., the probe sequence and the database sequence
  • Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications.
  • the terms "homology” and "identity" in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • One example of a useful algorithm is BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. Mol. Biol. 215:403-410 (1990), respectively.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0).
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873 (1993)).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001.
  • Sequence homology means that two polynucleotide sequences are homologous (i e. , on a nucleotide-by-nucleotide basis) over the window of comparison.
  • a percentage of sequence identity or homology is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence homology.
  • This substantial homology denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 60 percent sequence homology, typically at least 70 percent homology, often 80 to 90 percent sequence homology, and most commonly at least 99 percent sequence homology as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence homology is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Sequences having sufficient homology can the be further identified by any annotations contained in the database, including, for example, species and activity information. Accordingly, in a typical environmental sample, a pluraUty of nucleic acid sequences will be obtained, cloned, sequenced and corresponding homologous sequences from a database identified. This information provides a profile of the polynucleotides present in the sample, including one or more features associated with the polynucleotide including the organism and activity associated with that sequence or any polypeptide encoded by that sequence based on the database information.
  • fingerprint or “profile” refers to the fact that each sample will have associated with it a set of polynucleotides characteristic of the sample and the environment from which it was derived. Such a profile can include the amount and type of sequences present in the sample, as well as information regarding the potential activities encoded by the polynucleotides and the organisms from which polynucleotides were derived. This unique pattern is each sample's profile or fingerprint.
  • a particular cloned polynucleotide sequence once its identity or activity is determined or a suggested identity or activity is associated with the polynucleotide.
  • the desired clone if not already cloned into an expression vector, is ligated downstream of a regulatory control element (e.g., a promoter or enhancer) and cloned into a suitable host cell.
  • a regulatory control element e.g., a promoter or enhancer
  • expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, phosmids, bacterial artificial chromosomes, viral nucleic acid (e.g., vaccinia, adeno virus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus, yeast, and the like)
  • the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: ⁇ QE70, pQE60, pQE-9 (Qiagen), psiX174, pBluescript SK, pBluescript KS, pNH8A, pNH16a, pNH18A, pNH46A (Sfratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Sfratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in
  • the nucleic acid sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and tip.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • the expression vectors typically contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the nucleic acid sequence(s) selected, cloned and sequenced as hereinabove described can additionally be introduced into a suitable host to prepare a library which is screened for the desired biomolecule or bioactivity.
  • the selected nucleic acid is preferably already in a vector which includes appropriate control sequences whereby a selected nucleic acid encoding a biomolecule or bioactivity may be expressed, for detection of the desired activity.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • nucleic acid sequence is amplified by PCR reaction or similar reaction known to those of skill in the art.
  • amplification kits are available to carry out such amplification reactions.
  • the alignment algorithms and searchable database can be implemented in computer hardware, software or a combination thereof. Accordingly, the isolation, processing and identification of nucleic acid or polypeptide sequences can be implemented in an automated system.
  • a number of traditional assay system exist for measuring an enzymatic activity using multi-well plates.
  • existing screening technology usually relies on two-dimensional well (e.g., 96-, 384- and 1536-well ) plates.
  • the present invention also provides a capillary array-based approach of that has numerous advantages over well-based screening techniques, including the elimination of the need for fluid dispensers for dispensing fluids (e.g., reactants) into individual well reservoirs, and the reduced cost per array (e.g., glass capillaries are reusable) (see, for example, U.S. Patent Application Serial No. 09/444,112, filed November 22, 1999, which is incorporated herein by reference in its entirety).
  • the capillaries, capillary array and systems of the invention are particularly well suited for screening libraries for activity or biomolecules of interest including polynucleotides.
  • the screening for activity may be effected on individual expression clones or may be initially effected on a mixture of expression clones to ascertain whether or not the mixture has one or more specified activities. If the mixture has a specified activity, then the individual clones may be rescreened for such activity or for a more specific activity after collection from the capillary array.
  • DNA isolation DNA is isolated using the IsoQuick Procedure as per manufacture's instructions (Orca Research Inc., Bothell, WA). The isolated DNA can optionally be normalized according to Example 2 (below). Upon isolation, the DNA is sheared by pushing and pulling the DNA through a 25-gauge double-hub needle and a 1- cc syringe about 500 times. A small amount is run on a 0.8% agarose gel to make sure the majority of the DNA is in the desired size range (about 3-6kb).
  • the DNA is blunt-ended by mixing 45 ⁇ l of 10X Mung Bean Buffer, 2.0 ⁇ l Mung Bean Nuclease (1050 u/ ⁇ l) and water to a final volume of 405 ⁇ l. The mixture is incubated at 37 °C for 15 minutes. The mixture is phenol;chlorofonn extracted, followed by an additional chloroform extraction. One ml of ice cold ethanol is added to the final extract to precipitate the DNA. The DNA is precipitated for 10 minutes on ice. The DNA is removed by centrifugation in a microcentrifuge for 30 minutes. The pellet is washed with 1 ml of 70% ethanol and repelleted in the microcentrifuge. Following centrifugation, the DNA is dried and gently resuspended in 26 ⁇ l of TE buffer.
  • Methylase Buffer 0.5 ⁇ l SAM (32 mM), 5.0 ⁇ l EcoRI Methylase (40 u/ ⁇ l) and incubating at 37 °C for 1 hour.
  • the following can be added to the methylation reaction: 5.0 ⁇ l of 100 mM MgCl 2 , 8.0 ⁇ l of dNTP mix (2.5 mM of each dGTP, dATP, dTTP, dCTP), 4.0 ⁇ l of Klenow (5u/ ⁇ l). The mixture is then incubated at 12 °C for 30 minutes.
  • IX STE is added.
  • the mixture is phenol/chloroform extracted once followed by an additional chloroform extraction.
  • One ml of ice cold ethanol is added to the final extract to precipitate the DNA.
  • the DNA is precipitated for 10 minutes on ice.
  • the DNA is removed by centrifugation in a microcentrifuge for 30 minutes. The pellet is washed with 1 ml of 70% ethanol, repelleted in the microcentrifuge and allowed to dry for 10 minutes.
  • the DNA is ligated by gently resuspending the DNA in 8 ⁇ l EcoRI adapters (from Sfratagene's cDNA Synthesis Kit), 1.0 ⁇ l of 10 X ligation buffer, 1.0 ⁇ l of 10 mM rATP, 1.0 ⁇ l of T4 DNA Ligase (4Wu/ ⁇ l) and incubating at 4 °C for 2 days. The ligation reaction is terminated by heating for 30 minutes at 70 °C.
  • the adapter ends are phosphorylated by mixing the ligation reaction with 1.0 ⁇ l of 10X Ligation Buffer, 2.0 ⁇ l of 10 mM rATP, 6.0 ⁇ l of H 2 0, 1.0 ⁇ l of polynucleotide kinase (PNK), and incubating at 37 °C for 30 minutes. After incubating for 30 minutes, 31 ⁇ l of H 2 O and 5 ml of 10 X STE are added to the reaction and the sample is size fractionated on a Sephacryl S-500 spin column. The pooled fractions (1-3) are phenol/chloroform extracted once, followed by an additional chloroform extraction.
  • PNK polynucleotide kinase
  • the DNA is precipitated by the addition of ice cold ethanol on ice for 10 minutes.
  • the precipitate is pelleted by centrifugation in a microcentrifuge at high speed for 30 minutes.
  • the resulting pellet is washed with 1 ml 70% ethanol, repelleted by centrifugation and allowed to dry for 10 minutes.
  • the sample is resuspended in 10.5 ⁇ l TE buffer.
  • the sample is not plated, but is ligated directly to lambda arms as described above, except 2.5 ⁇ l of DNA and no water is used.
  • Sucrose Gradient (2.2 ml) Size Fractionation. Ligation is stopped by heating the sample to 65 °C for 10 minutes. The sample is gently loaded on a 2.2 ml sucrose gradient and centrifuged in a mini-ultracentrifuged 45k rpm at 20 °C for 4 hours (no brake). Fractions are collected by puncturing the bottom of the gradient tube with a 20 - gauge needle and allowing the sucrose to flow through the needle. The first 20 drops are collected in a Falcon 2059 tube, and then ten 1-drop fractions (labeled 1-10) are collected. Each drop is about 60 ⁇ l in volume. Five ⁇ l of each fraction are run on a 0.8% agarose gel to check the size.
  • Fractions 1-4 (about 10-1.5 kb) are pooled and, in a separate tube, fractions 5-7 (about 5-0.5 kb) are pooled.
  • One ml of ice cold ethanol is added to precipitate the DNA and then placed on ice for 10 minutes.
  • the precipitate is pelleted by centrifugation in a microcentrifuge at high speed for 30 minutes.
  • the pellets are washed by resuspending them in 1 ml of 70% ethanol and repelleting them by centrifugation in a microcentrifuge at high speed for 10 minutes, and then dried. Each pellet is then resuspended in 10 ⁇ l of TE buffer.
  • Test Ligation to Lambda Arms The assay is plated by spotting 0.5 ⁇ l of the sample on agarose containing ethidium bromide along with standards (DNA sample of known concentration) to get an approximate concentration. The samples are then viewed using UV light and the estimated concentration is compared to the standards.
  • the phage suspension is recovered by pouring the SM buffer off each plate into a 50 ml conical tube. About 3 ml of chloroform are added, shaken vigorously and incubated at room temperature for 15 minutes. The tubes are centrifuged at 2K rpm for 10 minutes to remove cell debris. The supernatant is poured into a sterile flask, 500 ⁇ l chloroform are added and stored at 4 °C.
  • One tube is inoculated with 10 ⁇ l of 10 "6 dilution (10 "5 ).
  • the other tube is inoculated with 1 ⁇ l of 10 "6 dilution (10 " ⁇ ), and incubated at 37 °C for 15 minutes.
  • the ZAP ⁇ library is excised to create the pBLUESCRIPT library according to manufacturer's protocols (Sfratagene).
  • the DNA library can be transformed into host cells (e.g., E. coli) to generate an expression library of clones.
  • host cells e.g., E. coli
  • Rf 1.3980
  • a syringe pump and fractionator (Brandel Model 186) are used to drive the gradient through an ISCO UA-5UV absorbance detector set to 280 nm. Peaks representing the DNA from the organisms present in an environmental sample are obtained. Eubacterial sequences can be detected by PCR amplification of DNA encoding rRNA from a 10 fold dilution of the E. coli peak using the following primers to amplify:
  • Reverse primer 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:5)
  • Recovered DNA is sheared or enzymatically digested to 3-6 kb fragments.
  • Lone-linker primers are ligated and the DNA is size-selected. Size-selected DNA is amplified by PCR, if necessary.
  • EDTA/0.1% SDS EDTA/0.1% SDS
  • the sample is overlaid with mineral oil and denatured by boiling for 10 minutes.
  • the sample is incubated at 68 °C for 12-36 hours.
  • Double-stranded DNA is separated from single-stranded DNA according to standard protocols (Sambrook, 1989) on hydroxyapatite at 60 °C.
  • the single-stranded DNA fraction is desalted and amplified by PCR. The process is repeated for several more rounds (up to 5 or more).
  • Plates of the library prepared as described in Example 1 are used to multiply inoculate a single plate containing 200 ⁇ l of LB Amp/Meth, glycerol in each well. This step is performed using the High Density Replicating Tool (HDRT) of the Beckman BIOMEK.RTM. with a 1% bleach, water, isopropanol, air-dry sterilization cycle between each inoculation.
  • the single plate is grown for 2h at 37 °C and is then used to inoculate two white 96-well Dynatech microtiter daughter plates containing 250 ⁇ l of LB Amp/Meth, glycerol in each well.
  • HDRT High Density Replicating Tool
  • the original single plate is incubated at 37 °C for 18h, then stored at -80 °C
  • the two condensed daughter plates are incubated at 37 °C also for 18 h.
  • the condensed daughter plates are then heated at 70 °C for 45 min. to kill the cells and inactivate the host E. coli enzymes.
  • a stock solution of 5 mg/mL morphourea phenylalanyl-7-amino-4-trifluoromethyl coumarin (MuPheAFC, the "substrate”) in DMSO is diluted to 600 ⁇ M with 50 mM pH 7.5 Hepes buffer containing 0.6 mg/mL of the detergent dodecyl maltoside.
  • the data will indicate whether one of the clones in a particular well is hydrolyzing the substrate.
  • the source library plates are thawed and the individual clones are used to singly inoculate a new plate containing LB Amp/Meth, glycerol. As above, the plate is incubated at 37 °C to grow the cells, heated at 70 °C to inactivate the host enzymes, and 50 ⁇ l of 600 ⁇ M MuPheAFC is added using the Biomek.
  • the enantioselectivity value, E, for the substrate is determined according to the equation below:
  • enantiomeric excess is dete ⁇ nined by either chiral high performance liquid chromatography (HPLC) or chiral capillary electrophoresis (CE). Assays are performed as follows: two hundred ⁇ l of the appropriate buffer is added to each well of a 96-well white microtiter plate, followed by 50 ⁇ l of partially or completely purified enzyme solution; 50 ⁇ l of substrate is added and the increase in fluorescence monitored versus time until 50% of the substrate is consumed or the reaction stops, whichever comes first.
  • HPLC high performance liquid chromatography
  • CE chiral capillary electrophoresis
  • the XL 1 -Red strain (Sfratagene) was transformed with genomic clone 27a3a (in plasmid pBluescript) encoding the alkaline phosphatase gene from the organism OC9a, an organism isolated from the surface of a whale bone, according to the manufacturer's protocol.
  • genomic clone 27a3a in plasmid pBluescript
  • a 5 ml culture of LB+0.1 mg/ml ampicillin was inoculated with 200 ⁇ l of the transformation and the culture was allowed to grow at 37 °C for 30 hours.
  • a miniprep was then performed on the culture, and the isolated DNA screened by transforming 2 ⁇ l of the resulting DNA into XL-1 Blue cells (Sfratagene) according to the manufacturer's protocol and following the assay procedure outlined below.
  • the mutated OC9a phosphatase took 10 minutes to develop color and the wild type enzyme took 30 minutes to develop color in the screening assay.
  • Transformed XL1 Blue cells were plated on LB/amp plates. The resulting colonies were lifted with Duralon UV (Sfratagene) or HATF (Millipore) membranes and lysed in chloroform vapors for 30 seconds. Cells were heat killed by incubating for 30 minutes at 85 °C. The filters were developed at room temperature in BCIP buffer and the fastest developing colonies ("positives") were selected for resfreaking the "positives" onto a BCIP plate (BCIP Buffer: 20 mm CAPS pH 9.0, 1 mm MgCl 2 , 0.01 mm ZnCl 2 , 0.1 mg/ml BCIP).
  • PCR was carried out by incubating 2 microliters dNTP's (10 mM Stocks); 10 microliters lOxPCR Buffer; 0.5 microliters Vector DNA-31G1A-100 nanograms; 20 microliters 3' Primer (100 pmol); 20 microliters 5' Primer (100 pmol); 16 microliters MnCl 4H 2 O (1.25 mM Stock); 24.5 microliters H 2 O; and 1 microliter Taq Polymerase (5.0 Units) in a total volume of 100 microliters.
  • the PCR cycle was: 95 °C 15 seconds; 58 °C 30 seconds; 72 °C 90 seconds; 25 cycles (10 minute extension at 72 °C -4 °C incubation).
  • Five microliters of the PCR product was run on a 1% agarose gel to check the reaction. Purify on a QIAQUICK column (Qiagen). Resuspend in 50 microliters H 2 O.
  • the resulting vector was transformed into M15pREP4 cells using electroporation. 100 or 200 microliters of the cells were plated onto LB amp meth kan plates, and grown overnight at 37 °C.
  • Beta-galactosidase was assayed by (1) Perform colony lifts using Millipore HATF membrane filters; (2) lyse colonies with chloroform vapor in 150 mm glass petri dishes; (3) transfer filters to 100 mm glass petri dishes containing a piece of Whatman 3MM filter paper saturated with Z buffer containing 1 mg/ml XGLU (After transferring filter bearing lysed colonies to the glass petri dish, maintain dish at room temperature); and (4) "Positives" were observed as blue spots on the filter membranes ("positives" are spots which appear early). A pasteur pipette (or glass capillary tube) was used to core blue spots on the filter membrane. Place the small filter disk in an Eppendorf tube containing 20 ⁇ l water.
  • the filter assay uses buffer Z (see recipe below) containing 1 mg/ml of the subsfrate 5-bromo-4-chloro- 3-indolyl-.beta.-o-glucopyranoside (XGLU) (Diagnostic Chemicals Limited or Sigma).
  • Z-Buffer (referenced in Miller, J. H. (1992) A Short Course in Bacterial Genetics, p. 445.) per liter:
  • the cell suspension was mixed with one volume of 1% molten Seaplaque LMP agarose (FMC) cooled to 40°C, and then immediately drawn into a 1 ml syringe.
  • FMC Seaplaque LMP agarose
  • the syringe was sealed with parafilm and placed on ice for 10 min.
  • the cell-containing agarose plug was extruded into 10 ml of Lysis Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 0.1M EDTA, 1% Sarkosyl, 0.2% sodium deoxycholate, 1 mg/ml lysozyme) and incubated at 37 °C for one hour.
  • Lysis Buffer 10 mM Tris pH 8.0, 50 mM NaCl, 0.1M EDTA, 1% Sarkosyl, 0.2% sodium deoxycholate, 1 mg/ml lysozyme
  • the agarose plug was then transferred to 40 mis of ESP Buffer (1% Sarkosyl, 1 mg/ml proteinase K, in 0.5M EDTA), and incubated at 55 °C for 16 hours. The solution was decanted and replaced with fresh ESP Buffer, and incubated at 55 °C for an additional hour. The agarose plugs were then placed in 50 mM EDTA and stored at 4 °C shipboard for the duration of the oceanographic cruise.
  • ESP Buffer 1% Sarkosyl, 1 mg/ml proteinase K, in 0.5M EDTA
  • the solution was then changed to 250 ⁇ l of the same buffer containing 4U of Sau3Al (NEB), equilibrated to 37 °C in a water bath, and then incubated on a rocking platform in a 37 °C incubator for 45 min.
  • the plug was transferred to a 1.5 ml microcentrifuge tube and incubated at 68 °C for 30 min to inactivate the enzyme and to melt the agarose.
  • the agarose was digested and the DNA dephosphorylased using
  • Vector arms were prepared from pFOSl as described (Kim et al, Stable propagation of cosmid sized human DNA inserts in an F factor based vector, Nucl. Acids Res., 20:10832-10835, 1992). Briefly, the plasmid was completely digested with Astll, dephosphorylated with HK phosphatase, and then digested with BamHI to generate two arms, each of which contained a cos site in the proper orientation for cloning and packaging ligated DNA between 35-45 kbp. The partially digested picoplankton DNA was ligated overnight to the PFOSl arms in a 15 ⁇ l ligation reaction containing 25 ng each of vector and insert and 1U of T4 DNA ligase (Boehringer-
  • the ligated DNA in four microliters of this reaction was in vitro packaged using the Gigapack XL packaging system (Sfratagene), the fosmid particles transfected to E. coli strain DH10B (BRL), and the cells spread onto LB cm ⁇ 5 plates.
  • the resultant fosmid clones were picked into 96-well microliter dishes containing LB cm ⁇ 5 supplemented with 7% glycerol.
  • Recombinant fosmids, each containing ca. 40 kb of picoplankton DNA insert yielded a library of 3.552 fosmid clones, containing approximately 1.4xl0 8 base pairs of cloned DNA. All of the clones examined contained inserts ranging from 38 to 42 kbp. This library was stored frozen at -80 °C for later analysis.
  • One microgram samples of template DNA are obtained and treated with U.V light to cause the formation of dimers, including TT dimers, particularly purine dimers.
  • U.V exposure is limited so that only a few photoproducts are generated per gene on the template DNA sample.
  • Multiple samples are treated with U.V. light for varying periods of time to obtain template DNA samples with varying numbers of dimers from U.V. exposure.
  • a random priming kit which utilizes a non-proofreading polymease for example, Prime-It II Random Primer Labeling kit by Sfratagene Cloning Systems
  • a non-proofreading polymease for example, Prime-It II Random Primer Labeling kit by Sfratagene Cloning Systems
  • the priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers.
  • the dimers formed by U.V exposure serve as a roadblock for the extension by the non-proofreading polymerase.
  • a pool of random size polynucleotides is present after extension with the random primers is finished.
  • Polynucleotides of interest which are generated according to Example 1 are gel isolated on a 1.5% agarose gel. Polynucleotides in the 100-300 bp range are cut out of the gel and 3 volumes of 6 M Nal is added to the gel slice. The mixture is incubated at 50 °C for 10 minutes and 10 ⁇ l of glass milk (Bio 101) is added. The mixture is spun for 1 minute and the supernatant is decanted. The pellet is washed with 500 ⁇ l of Column Wash (Column Wash is 50% ethanol, lOmM Tris-HCl pH 7.5, 100 mM NaCl and 2.5 mM EDTA) and spin for 1 minute, after which the supernatant is decanted. The washing, spinning and decanting steps are then repeated. The glass milk pellet is resuspended in 20 ⁇ l of H 2 O and spun for 1 minute. DNA remains in the aqueous phase.
  • the 100-300 bp polynucleotides obtained in Example 2 are recombined in an annealing mixture (0.2 mM each dNTP, 2.2 mM MgCl 2 , 50 mM KC1, 10 mM Tris- HCl ph 8.8, 0.1% Triton X-100, 0.3 ⁇ ; Taq DNA polymerase, 50 ⁇ l total volume) without adding primers.
  • a Robocycler by Sfratagene was used for the annealing step with the following program: 95 °C for 30 seconds, 25-50 cycles of [95 °C for 30 seconds, 50 - 60 °C (preferably 58 °C) for 30 seconds, and 72 °C for 30 seconds] and 5 minutes at 72 °C.
  • the 100-300 bp polynucleotides combine to yield double- stranded polynucleotides having a longer sequence.
  • the polynucleotides of Example 3 are separated and polypeptides are expressed therefrom.
  • the original template DNA is utilized as a comparative control by obtaining comparative polypeptides therefrom.
  • the polypeptides obtained from the shuffled polynucleotides of Example 3 are screened for the activity of the polypeptides obtained from the original template and compared with the activity levels of the control.
  • the shuffled polynucleotides coding for interesting polypeptides discovered during screening are compared further for secondary desirable traits. Some shuffled polynucleotides corresponding to less interesting screened polypeptides are subjected to reshuffling.
  • Site-Saturation Mutagenesis To accomplish site-saturation mutagenesis every residue (316) of a dehalogenase enzyme was converted into all 20 amino acids by site directed mutagenesis using 32-fold degenerate oligonucleotide primers, as follows:
  • a culture of the dehalogenase expression construct was grown and a preparation of the plasmid was made. Primers were made to randomize each codon - they have the common structure X 2 oNN(G/T)X 20 ; where X 20 represents a 20-base sequence with sufficient homology to bind the template, and NN(G/T) is a 32-fold degenerate 3-base sequence that encodes all 20 amino acids.
  • this invention provides for the use of, in addition to an N,N,G/T or identically NN(G/T) cassette, any set of codons that is serviceable for introducing all 19 amino acid substitutions including (but not limited to) a 32-fold degenerate NN(G/C) cassette, a 64-fold degenerate NNN cassette, a 48-fold NN(A/C/G) cassette, a 48-fold NN(A/G/T) cassette, any non- degenerate set of 19 codons that is serviceable for introducing all 19 amino acid substitutions, and any set of 20 codons is serviceable for introducing all 19 amino acid substitutions.
  • this invention provides that a subset of the 20 amino acids is introduced at each of a set of codon positions; an example of such a subset can comprise amino acid(s) selected from a subset based on a grouping scheme, which grouping scheme can be, by way of non-limiting exemplification, the following grouping scheme with 8 subsets:
  • a reaction mix of 25 ul was prepared containing ⁇ 50 ng of plasmid template, 125 ng of each primer, IX native Pfu buffer, 200 uM each dNTP and 2.5 U native Pfu DNA polymerase
  • reaction mix was digested with 10 U of Dpnl at 37°C for 1 hour to digest the methylated template DNA
  • Vaccina Topoisomerase I (Invitrogen, Carlsbad, CA) was added to
  • the topogated PCR product was cloned into the vector pASK3 (5 ⁇ l, ca. 200 ng in
  • This invention provides that a desirable property to be generated by directed evolution is exemplified in a limiting fashion by an improved residual activity (e.g. an enzymatic activity, an immunoreactivity, an antibiotic activity, etc.) of a molecule upon subjection to altered environment, including what may be considered a harsh enviroment, for a specified time.
  • an improved residual activity e.g. an enzymatic activity, an immunoreactivity, an antibiotic activity, etc.
  • Such a harsh environment may comprise any combination of the following (iteratively or not, and in any order or permutation): an elevated temperature (including a temperature that may cause denaturation of a working enzyme), a decreased temperature, an elevated salinity, a decreased salinity, an elevated pH, a decreased pH, an elevated pressure, a decreased pressure, and an change in exposure to a radiation source (including uv radiation, visible light, as well as the entire electromagnetic spectrum).
  • a desirable property such as an improved residual activity can be an improved activity (e.g. higher activity) after a certain period of maintenance in a harsh environment (e.g. higher temperature, lower temperature, higher pH, lower pH, higher salinity, lower salinity, increased organic solvent presence, lower organic solvent presence, etc. ) can comprise a selection criterion for a screening or selection step following the application of a directed evolution step, such as, but not limited to, an end-selection-based step, an exonuclease treatment- based step, a site-saturation mutagenesis-based step, or a synthetic ligation reassembly-based step.
  • a directed evolution step such as, but not limited to, an end-selection-based step, an exonuclease treatment- based step, a site-saturation mutagenesis-based step, or a synthetic ligation reassembly-based step.
  • a desirable property such as an improved residual activity an improved activity (e.g. higher activity) after exposure to, followed by removal from, a harsh environment (e.g. higher temperature, lower temperature, higher pH, lower pH, higher salinity, lower salinity, increased organic solvent presence, lower organic solvent presence, etc. )
  • a harsh environment e.g. higher temperature, lower temperature, higher pH, lower pH, higher salinity, lower salinity, increased organic solvent presence, lower organic solvent presence, etc.
  • a directed evolution step such as, but not limited to, an end-selection-based step, an exonuclease treatment-based step, a site-saturation mutagenesis-based step, or a synthetic ligation reassembly-based step.
  • the residual activity results from an increased ability of a molecule to regain, after subjection to denaturing conditions, an active 3- dimensional form (including the original 3 -dimensional form, a 3 -dimensional form that is similar to the original 3 -dimensional form, or a 3 -dimensional form that is different than the original 3 -dimensional form).
  • This ability to re-fold is sometimes referred to as "reversibility of denaturation" or "re-naturation ability" or
  • the robot made 2 assay plates from each parent growth plate consisting of 0.02 mL cell suspension. 3. One assay plate was placed at room temperature and the other at elevated temperature (initial screen used 55°C) for a period of time (initially 30 minutes).
  • Thermal stability was assessed by incubating the enzyme at the elevated temperature (55°C and 80°C) for some period of time and activity assay at 30°C. Initial rates were plotted vs. time at the higher temperature.
  • the enzyme was in 50 mM Tris/SO 4 pH 7.8 for both the incubation and the assay.
  • Product (Cf) was detected by a standard method using Fe(NO 3 ) and HgSCN.
  • Dhla 20F12 was used as the de facto wild type.
  • the apparent half-life (Tj /2 ) was calculated by fitting the data to an exponential decay function.
  • Enzyme progress curves for Dhla 5 and 20F12 at 30°C and 55°C see Figure 14).
  • the reaction was performed in 50 mM Tris/SO 2" , pH 7.8 with 10 mM TCP using purified protein. Equal amounts of protein were used in each assay.
  • Product (CF) concentration was determined at variable times using a standard Cl " assay. Dhla 5 appears to have the same specific activity as 20F12 at both 30°C and 55°C but the enzyme continues to turn over after 20F12 is thermally inactivated.

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Abstract

L'invention concerne une méthode permettant de cribler des bibliothèques génétiques dérivées d'une population mixte d'organismes afin de détecter une bioactivité d'une biomolécule d'intérêt. La population mixte d'organismes peut être une population cultivée ou une population non cultivée issue, par exemple, de l'environnement. L'invention concerne également des méthodes permettant de cribler des isolats ou des populations enrichies d'organismes, lesquels isolats comprennent une population qui est spatialement ou temporellement hiérarchisée, et qui fait partie, par exemple, d'une espèce, d'une genre, d'une famille ou d'une classe particulières d'organismes. Des clones identifiés contenant une biomolécule ou présentant une bioactivité d'intérêt peuvent être panachés ou l'ADN contenu dans ledit clone peut être panaché afin de créer de nouvelles biomolécules ou bioactivités d'intérêt.
PCT/US2001/029712 2000-09-15 2001-09-17 Criblage combinatoire de populations mixtes d'organismes WO2002022810A2 (fr)

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CA002391626A CA2391626A1 (fr) 2000-09-15 2001-09-17 Criblage combinatoire de populations mixtes d'organismes
JP2002527252A JP2004524811A (ja) 2000-09-15 2001-09-17 有機体の混合集団の組み合わせスクリーニング
EP01971309A EP1319068A2 (fr) 2000-09-15 2001-09-17 Criblage combinatoire de populations mixtes d'organismes
AU91208/01A AU9120801A (en) 2000-09-15 2001-09-17 Combinatorial screening of mixed populations of organisms

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US09/663,620 US7018793B1 (en) 1995-12-07 2000-09-15 Combinatorial screening of mixed populations of organisms
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1507870A2 (fr) * 2000-12-01 2005-02-23 Diversa Corporation Enzymes dotes d'une activite de deshalogenase et leurs methodes d'utilisation
CN102590528A (zh) * 2012-01-18 2012-07-18 厦门大学 一种蛋白相互作用的检测方法
CN106124471A (zh) * 2016-07-19 2016-11-16 天津大学 时域荧光寿命成像结构及寿命测试获取方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006235875B8 (en) * 2000-12-01 2011-04-14 Basf Enzymes Llc Enzymes having dehalogenase activity and methods of use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020078A1 (fr) * 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
WO1998032845A1 (fr) * 1997-01-24 1998-07-30 Bioinvent International Ab Procede d'evolution moleculaire in vitro de la fonction proteique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020078A1 (fr) * 1995-11-30 1997-06-05 Maxygen, Inc. Procede d'elaboration de polynucleotides presentant des caracteristiques desirees par selection iterative et recombinaison
WO1998032845A1 (fr) * 1997-01-24 1998-07-30 Bioinvent International Ab Procede d'evolution moleculaire in vitro de la fonction proteique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHULTZ S ET AL: "Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 83, no. 6, March 1986 (1986-03), pages 1588-1592, XP002147196 ISSN: 0027-8424 *
STEMMER W P C: "Rapid evolution of a protein in vitro by DNA shuffling" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, vol. 370, 4 August 1994 (1994-08-04), pages 389-391, XP002082182 ISSN: 0028-0836 cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1507870A2 (fr) * 2000-12-01 2005-02-23 Diversa Corporation Enzymes dotes d'une activite de deshalogenase et leurs methodes d'utilisation
EP1507870A4 (fr) * 2000-12-01 2005-11-09 Diversa Corp Enzymes dotes d'une activite de deshalogenase et leurs methodes d'utilisation
US7078504B2 (en) 2000-12-01 2006-07-18 Diversa Corporation Enzymes having dehalogenase activity and methods of use thereof
US7671189B2 (en) 2000-12-01 2010-03-02 Verenium Corporation Enzymes having dehalogenase activity and methods of use thereof
US8153411B2 (en) 2000-12-01 2012-04-10 Verenium Corporation Dehalogenases, nucleic acids encoding them and methods for making and using them
US9453211B2 (en) 2000-12-01 2016-09-27 Basf Enzymes Llc Dehalogenases, nucleic acids encoding them and methods for making and using them
CN102590528A (zh) * 2012-01-18 2012-07-18 厦门大学 一种蛋白相互作用的检测方法
CN106124471A (zh) * 2016-07-19 2016-11-16 天津大学 时域荧光寿命成像结构及寿命测试获取方法
CN106124471B (zh) * 2016-07-19 2019-04-16 天津大学 时域荧光寿命成像结构及寿命测试获取方法

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CA2391626A1 (fr) 2002-03-21

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