JP2003509010A - Peptide screening method - Google Patents

Abstract

  (57) [Summary] A method for screening a nucleotide sequence encoding an antimicrobial peptide, comprising: (a) acting on an inducible promoter to express a peptide having less than 100 amino acid residues optionally linked to a signal peptide. Ligating the plasmid with the above pool of nucleotide sequences ligated in the following manner: (b) transforming host cells susceptible to the peptide with the ligated plasmid; and (c) selecting live cells. Screening said transformed host cells, (d) culturing said living cells in the presence of an inducer so as to induce expression of said nucleotide sequence, and (e) said induction in cell growth. Selecting a cell according to the action of the substance, and encoding the peptide encoding the peptide from the selected cell; Concerning the method comprising recovering the sequence. Emphasis is placed on DNA shuffling as a method for preparing nucleotide pools.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a method of screening a pool of nucleotide sequences for selecting nucleotide sequences that encode peptides.

BACKGROUND OF THE INVENTION Various bioactive peptides are known to kill or inhibit the growth of target cells, such as antimicrobial enzymes, antitumor peptides, and antimicrobial peptides.
Improved screening methods for those peptides are desirable for the development of new bioactive peptides.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for identifying new or improved existing genes encoding bioactive peptides capable of killing target cells or inhibiting their proliferation. is there. The present invention provides a suicide expression system for the above peptides.
A side expression system (SES) was developed. The S
The principle of ES is to produce a peptide-encoding library in cells, induce expression of individual peptides, and by virtue of their ability to kill or inhibit growth of host cells as a result of said peptide synthesis. To select / identify the coding sequence. The sequential process of peptide induction, selection, plasmid amplification, and mutagenesis can be used to identify peptides with improved bioactivity.
However, the unprotected or scaffolding peptide is
It is required to protect the active peptide from digestion within the cell. The peptides will be required for recovery and purification of the peptides, for example, not to identify the nucleotide sequences encoding the active peptides described in the present invention.

Accordingly, the present invention is a method of sequencing a pool of nucleotide sequences for selecting a nucleotide sequence encoding a peptide, comprising: a) less than 100 amino acid residues optionally linked to a signal peptide. A plasmid is ligated to the above-mentioned nucleotide sequence pool operably linked to an inducible promoter so as to express a peptide which is an enzyme or a mature peptide having b. In the presence of an inducer, c) screening the transformed host cells to select live cells, and d) inducing the expression of the nucleotide sequence, Culturing the above-mentioned living cells, and e) determining the effect of the above-mentioned inducer on cell proliferation. What cells is selected, and f) recovering the nucleotide sequence encoding the peptide from the selected cell to provide the method comprising. The principle of the present suicide expression system (SES) is to create a peptide library in microorganisms, induce the expression of said individual peptides, and kill or severely inhibit their growth as a result of sudden peptide synthesis. It depends on whether or not cells are selected / identified.

For the identification of new genes encoding antibacterial activity, putatively, a library of genes harboring antibacterial activity was cloned into relevant plasmids, inducing synthesis, growth-inhibited or killed. Bacteria were identified and the corresponding genes were sequenced and analyzed.

For the identification of peptide variants with enhanced bioactivity, a library of existing peptide mutants is generated and introduced into the target organism. The sequential process of peptide induction (gradually using smaller amounts of inducer), selection, plasmid amplification, and shuffling / mutagenesis will allow identification of peptides with improved bioactivity.

DETAILED DESCRIPTION OF THE INVENTION Peptides The methods of the invention can be used to screen peptides for their bioactivity, ie their ability to kill target cells or inhibit their proliferation. Thus, the peptide may be a peptide compound that interacts / binds / inactivates the original cellular target. The peptide of interest is an antimicrobial enzyme or a short peptide (less than 100 amino acid residues), such as an antimicrobial peptide (anti-micro).
It can be a bial peptide (AMP) or an anti-tumor peptide.

The antimicrobial enzyme can be, for example, muramidase, lysozyme, protease, lipase, phospholipase, chitinase, glucanase, cellulase, peroxidase or laccase. Alternatively, conventional antibiotics such as a consortium of enzymes that synthesize polyketides or penicillins may be utilized.

The antimicrobial peptide (AMP) can be a membrane active antimicrobial peptide or an antimicrobial peptide that acts / interacts with intracellular targets, eg, binds to cellular DNA. The A
MPs are relatively short peptides generally consisting of less than 100 amino acid residues, typically 20-80 residues. The antimicrobial peptide has a bactericidal and / or fungicidal action and may also have an antiviral or antitumor action. It generally has a slight cytotoxic effect on normal mammalian cells.

The antimicrobial peptides are generally highly cationic and hydrophobic. It typically contains some arginine and lysine, and may not contain glutamic acid or aspartic acid alone. It generally contains the most hydrophobic residues.
The peptides generally have an amphipathic structure with one surface having a high positive charge and another hydrophobic surface.

The bioactive peptide and the nucleotide sequence encoding it may be provided by plants, invertebrates, insects, amphibians and mammals, or microorganisms such as bacteria and fungi.

The antimicrobial peptide acts on the cell membrane of the target microorganism, usually in a direction parallel to the membrane, for example through nonspecific binding to the membrane, and interacts only with one side of the bilayer membrane.

The antimicrobial peptides are typically divided into five major categories: α-helical, (defensin-like) cystine-rich, β-sheet, peptides with a unique composition consisting of regular amino acids, and rare It has a structure belonging to one of the peptides containing modified amino acids.

Examples of alpha-helical peptides are: Magainin 1 and 2; Cecropins A, B, and P1; CAP18; Andropin; Clavanin A and AK; Styelin. ) D and C; and Buforin II
Is. An example of a cystine-rich peptide is α-defensin (Defens
in) HNP-1 (human neutrophil peptide), HNP-2, and HNP-3; β-
Defensin-12; Drosomycin; γ1-Prothionin; and insect defensin A. Examples of β-sheet peptides are lactoferricin B;
Tachyplesin I; and Protegrins
rin) PG1-5. Examples of peptides with a unique structure are Indolicidin; PR-39; Bactenicin.
n) Bac5 and Bac7; and Histatin 5.
Examples of peptides with unique amino acids are Nisin; Gramicidin A; and Alamethicin.

[0015] Other examples are, it is an anti-fungal peptide from Aspergillus Giganteusu (Aspergillus g iganteus).

In a preferred embodiment said expressed peptide does not have any protective scaffold protein.

Nucleotide Sequence Pool Commercially available antimicrobial peptides, such as antibiotics or antibacterial agents, rely on their potency, species specificity, and ability to perform under unique conditions. In general, these conditions are quite different from the conditions under which the peptides originally evolved. Most antimicrobial peptides are, for example, to target a wide range of different microorganisms present at the same time, to work within a range of physiological salts, to evade the human immune system, or to eliminate the mammalian circulatory system. It was never evolved to compete.

For a given antimicrobial peptide, this dilemma creates random variants of the parent sequence, either by logical modification based on knowledge of the peptide or by inducing further evolution of the peptide, Then it can in principle be solved by either selecting mutants in which the combination of required properties is found. Directed evolution, an iterative process that explores vast regions of sequence space to yield muteins and peptides with unique desirable characteristics, combined with powerful high-throughput assays, encodes for natural or modified genes. It allows large libraries of selected antimicrobial peptides to be produced and evaluated for identification of key candidates with the required characteristics. These approaches are currently
It has been adopted widely by academia and industry alike at unprecedented rates to produce new proteins based on activity.

The nucleotide sequence encoding the bioactive peptide can be obtained from chromosomal DNA from one of the above mentioned source organisms and / or can be chemically synthesized. The nucleotide sequence can also be a cDNA derived from the source organism.

The sequencing method of the present invention can be used for the development of peptides having improved physiological activity. Thus, starting from a known gene encoding a bioactive peptide, a DNA pool can be obtained, for example by random mutagenesis to create a mutation library, by gene shuffling, or by synthesis of degenerate genes. .

The shuffled sequences can be related sequences from different organisms (so-called “family shuffling”) or they can include parent sequences and variants thereof.

In a preferred embodiment of the present invention random mutagenesis is performed, for example, by Stemmer (Ste
mmer, 1994. Proc. Natl. Acad. Sci. USA, 91: 10747-10751; Stemmer, 1994.
Nature 370: 389-391) and Crameri, A., et al., 1997. Nature Biotechnology
15: 436-438 by shuffling homologous DNA in vitro, all of which are incorporated by reference.

The method comprises homologous DNA by using in vitro PCR technology.
Regarding array shuffling. Active recombinant genes containing shuffled DNA sequences are selected from DNA libraries based on the enhanced action of the expressed proteins.

The previous method is also described in WO 95/22625, which is hereby incorporated by reference for the method of shuffling homologous DNA sequences. One important step of the method is to cleave the homologous template duplex polynucleotide into a random fragment of the required size, after which the fragment is homologously reassociated with the full length gene.

In another preferred embodiment, random mutagenesis is performed by constructing a library of recombinant homologous polynucleotides from several different input template DNAs and primers due to template shifts induced during in vitro DNA synthesis. Described method of DNA shuffling, WO98 / 41653, incorporated herein by reference.
It is achieved by the method described in. In this respect, in particular, WO98 / 4165
The special version of this in vitro recombination through template shift induced during DNA synthesis, described in 3, is preferred. Short here (> 5 nucleotides)
Random DNA primers are utilized for the random initiation of DNA synthesis on the mutated DNA template to be combined.

Special care is contemplated when using each of the above methods of generating and merging sequence diversity for the gene encoding the small size antimicrobial peptide. Since most shuffling methods rely on flanking a sufficient number of identical base pairs (20-100 base pairs) to the recombination mutation, mutations in the short gene are merged by the method described above. That is technically difficult.

Therefore, another form of induced evolution applies to short genes. About 5
In a preferred embodiment involving the merging of variants of a peptide given less than 0 amino acids, one degenerate DNA primer harboring all the required variants will be synthesized. At the given position in this degenerate primer, there should be both mutated nucleotides in addition to wild type nucleotides. The frequency of mutations from wild type to mutated nucleotides can be adjusted to the optimal state contemplated; rules and considerations are known in the art. 1 for all required mutants
By including in one primer, the required sequence could be completely sampled. This method allows sampling and merging of all required mutants regardless of how similar they are to the original gene sequence.

If a peptide having more than about 50 amino acids is used, two or more independent,
And degenerate primers will be used. This is due to the limitation experienced during DNA primer synthesis, in that only nucleotide DNA primers of less than about 180-200 can be synthesized at any one time.

In another embodiment in which a peptide of more than about 50 amino acids is used, the sequence diversity combined (the individual variants) is combined in a short oligonucleotide of 20 to 30 base pairs in length. Can be housed individually. In this approach, a specific oligo DNA is used for each mutation that will be included in the library. The mutation should preferentially be located in the center of the short oligo DNA to optimize annealing. Spiking some or many of these short oligo DNAs in a PCR reaction using the wild-type peptide gene as the main force of this amplification will allow the merging of the required mutants. By varying the amount of the individual oligo DNAs that are combined, the required ratio of individual variants to wild type can be created. This method does not allow efficient merging with nearby flanking mutants, as approximately 10 base pairs on either end of the sequence are required to be mismatched.

The suicide expression system is not limited to the identification of improved variants of the existing and already characterized peptides. New genes may also be identified that encode peptides that affect the growth of a given host cell. A library of cDNA or randomly expressed whole chromosomal DNA fragments can be used as a starting material in the suicide expression system and cloned.

Host Cell The host cell must be sensitive to peptides, enzymes or secondary metabolites of interest.

Thus, for screening for antimicrobial peptides or enzymes, the host cell may be a bacterium, for example Escherichia coli ( E. coli ) or B. Subtilis (B. subtilis) such can be a Bacillus (Bacillus) or fungal cells, can be a yeast such as, for example, Aspergillus (Aspergillus) such as filamentous fungi or Saccharomyces genus (Saccharomyces) or Candida (Candida). It is preferred to use host cells which are related to the target microorganism against which the antimicrobial peptide is intended.

For screening for anti-tumor peptides, the host cells are preferably mammalian cells, especially tumor cells.

Desirably, the host cell is capable of transporting the inducer across a membrane, preferably without metabolizing or degrading them. This is advantageous as an expression test as inducer levels are constant inside the cells and do not decrease over time. This may be achieved by choosing a "gratuitous" inducer or by deleting one or more genes required for the metabolism of said inducer.

The host cell must be chosen in order to be able to express the antimicrobial peptide. Therefore, fungal cells are preferred for peptides with disulfide bridges, such as the cystine-rich peptides mentioned above.

Plasmid The plasmid must be replicable in the host organism and capable of expressing the bioactive peptide (and optionally the signal peptide) under the control of the inducible promoter. It will generally include a selectable marker, such as an antibiotic marker. Many of the above plasmids are known in the art.

Ligation A plasmid is ligated to the pool of nucleotide sequences operably linked to an inducible promoter so that the peptide of interest optionally linked to a signal peptide is expressed. The short peptide or enzyme of interest may be expressed without any extension (other than the additional signal peptide) or with a short extension of, for example, 1-5 amino acids. Expression in the form of a fusion protein is neither preferred nor necessary.

Inducible promoters and attractants The plasmids used according to the invention must contain an inducible promoter which regulates the expression of the inserted nucleotide sequence coding for said peptide. It is advantageous for the application of the SES that it allows the complete termination of the synthesis of the encoded bioactive peptide. Furthermore, the induction of the encoded bioactive peptide must be significant, since the peptide is inherently unstable in the cytoplasm of the microorganism and is easily degraded. The inducible promoter used in this example is regulated both positively and negatively by two proteins. In the presence of the inducer, expression from the promoter is initiated, and in the absence of inducer very low levels of expression from the promoter occur. Non-induced levels are even further suppressed by growth in the presence of secondary metabolites. By altering the activity of the two modulators, the expression level of the protein can be engineered for optimal toxicity with respect to potential toxicity or the original gene.

The promoter is regulated by the presence of a lactose inducer or by the synthetic indigestible lactose derivative IPTG, Taguchi S., Nakagawa K.
., Maeno M. and Momose H .; "In Vivo Monitoring System for Structure-Func
tion Relationship Analysis of the antibacterial peptide Apidaecin "; Appl
The Lac promoter described in ied and Environmental Microbiology, 1994, pp.3566-3572. Other inducible promoters are known in the art, such as the tryptophan-inducible trp promoter or the galactose-inducible E. coli. E. coli gal promoter, S. G of cerevisiae
all promoter, Pichia pastoris
AOX1 promoter, pMT (metallothionein) promoter of Drosophila (Drosophila), MMTV LTR on the expression of mammalian, pV of
gRXR or pIND promoter. Using an inducer that is not metabolized or digested in the cell offers the advantage that the inducer concentration can be kept constant throughout the screening process. However, a drawback of the Lac promoter is that it cannot be stopped completely due to the absence of the inducer. The promoter is also the pBAD promoter used in this example (see below). This promoter is driven by inter alia degradable arabinose inducers. However, in order to achieve the advantage of having a certain level of inducer mentioned above, the ability of the host cell to digest arabinose was determined from the host cell chromosome by a suitable gene (genotype description is found in this example). Can be)
Could be eliminated by deleting However, an important consideration regarding the selection of a suitable promoter is that it is desirable that the corresponding inducer be capable of penetrating the cell membrane to provide access to the promoter.

The pBAD promoter is regulated both positively and negatively by two proteins, AraC and cAMP-CRP. Expression from the promoter is initiated in the presence of arabinose, while very low levels of expression occur from the promoter even in the absence of arabinose.

Uninduced levels are even further suppressed by growth in the presence of glucose. Glucose serves to reduce cAMP levels, which similarly reduces binding of cAMP-CRP to the promoter region of pBAD. When cAMP levels are lowered, transcriptional activity is reduced. This is ideal if the peptide of interest is excessively growth inhibitory or toxic to the host. In short, by altering the activity of the two modulators, the expression level of the protein can be manipulated to optimize expression of the potential toxicity or native gene.

Signal Peptide The DNA sequence encoding the signal peptide is optionally downstream of the inducible promoter of the plasmid and is described above such that the bioactive peptide is expressed with the signal peptide bound thereto. It can be inserted upstream of the sequence encoding the bioactive peptide. A suitable signal for a given host cell
Peptides can be selected according to principles in the art.

Generally, the peptide of interest first attacks or permeates the target cell from the outside, and in many cases, the success of the SES requires that the peptide be carried out of the cell. To be In general, bioactive peptides cross the cell membrane and are secreted, for example, in the case of Gram-negative prokaryotes, into the periplasmic space, from where they interact with their cellular targets, such as cell membranes, membrane components or periplasmic spaces. Or is allowed to diffuse further through the outer cell membrane.

Where the peptide of interest can exert its action when expressed and retained intracellularly, for example, a peptide that binds to the DNA of the cell or a peptide that does not depend on its ability to penetrate the membrane or a peptide that has an intracellular target In the case of, the signal peptide may be omitted. One example is the proline-arginine rich peptide family Bac5, Bac7, and PR39, which are suggested in the literature to interact and sequester nucleic acids.

Screening Step As described above, the present invention is a method for screening a nucleotide sequence pool for selecting a nucleotide sequence encoding an antimicrobial peptide that acts on the cell membrane, cell wall or DNA of a target microorganism, comprising the steps of: (A) the above nucleotide sequence operably linked to an inducible promoter to express a peptide which is an enzyme or mutant peptide having less than 100 amino acid residues linked to a signal peptide. The pool and plasmid are ligated, (b) the host cell that is sensitive to the peptide is transformed with the ligated plasmid, and (c) the transformed host cell is selected to select live cells. And (d) induce expression of the above nucleotide sequence Sea urchin, in the presence of inducer,
Culturing said living cells, (e) selecting cells according to the effect of said inducer on cell proliferation, and (f) recovering the nucleotide sequence encoding said peptide from said selected cells, Including the above methods.

Certain preparatory steps are required prior to step (a) of the screening process. The host is required to discover peptides that kill and / or inhibit growth, and it is desirable to select a suitable plasmid compatible with that host. The nucleotide sequence pool provided, for example, by mutation of a known sequence encoding a known antimicrobial peptide, is preferably prepared by conventional methods as described above.

In step (a) the library is ligated into a suitable plasmid by conventional methods and transformed into the host cell culture (step (b
)).

Step (c) is a first screening or selection step, which separates live cells from cells that have been killed and / or become growth-inhibited during the transformation process. . This step is an important step because the ultimate purpose of the screening step is to identify cells that have been killed and / or growth inhibited by the induced expression of inserted nucleotide sequences that produce antimicrobial peptides. Because it is. Cells that have been killed and / or inhibited prior to the screening step, etc. without the antimicrobial peptide will generate a false active response in the screen if they were not separated from the live cells. In step (d) an inducer is introduced into the living cells that have been cultured to induce the expression of the nucleotide sequences contained in the insertion plasmid from the library. Since the peptide is produced by transcription, the host cell will be killed and / or growth-inhibited when the peptide has an antibacterial effect on the host. In a preferred embodiment, killed and / or growth-inhibited host cells are selected. By selection of killed and / or growth-inhibited host cells, cells containing the nucleotide sequence encoding the peptide having antibacterial activity can be isolated. Another level higher inducer concentration can be used in parallel to achieve a graded response and to lock in different antimicrobial effects or effects. Host cells which become killed and / or become growth-inhibited in step (e) are selected and influenced by peptides expressed from the inserted plasmid nucleotide sequences in the form of transformation of said host cells. Separated from no host cells. It selects, for example, only those cells that are strongly affected by the induced expression of peptides that are affected by low concentrations of inducer, or are within the intended range of screening and / or existing knowledge of nucleotide sequence pools or libraries. Depending on, all affected cells can be selected.

Criteria for selecting cells can be chosen for each, eg maximum inducer concentration is
Defined to select only those cells that are affected by the presence of an inducer below this inducer concentration, and / or reduced levels using a stepwise decreasing concentration of inducer to a replica of the transformed host cell. Transcriptional induction of E. coli would allow the isolation of peptides with increased bioactivity (Fig. 4).

The inserted nucleotide sequence may be recovered by conventional methods from the selected host cell identified as encoding a bioactive peptide. The nucleotide sequence may be amplified by conventional methods, eg PCR amplification. The nucleotide sequence identified and amplified from here is inserted into the production host,
And peptides consistent with those identified can be produced in large quantities according to known methods in which the peptide is expressed through fusion to a larger polypeptide that can be subsequently exported by the host cell. The polypeptide protects the peptide of interest from digestion in the cell and its inactivity by the host cell enzyme, and / or the host cell expressed peptide and the peptide are incorporated into the polypeptide. It has a function of reducing the action of the peptide in the host cell so that the growth and the expression of the peptide can be continued without being strongly influenced by the action that would otherwise occur. The confirmed and amplified nucleotide sequence encoding said peptide can also be mutated as described previously, eg by random mutagenesis, by gene shuffling or by synthesis of a degenerate gene. These newly mutated nucleotide sequences are then used to confirm the nucleotide sequences encoding the novel peptides with improved action,
It can be screened again according to steps (a) to (f), eg by lowering the inducer concentration in a later screen.

The screening or isolation step is, in a particular embodiment, carried out by utilizing a conventional plate assay to streak the transformed host cells onto plates containing nutrient medium and optionally antibiotics. If the transformation plasmid contains a gene for resistance to the antibiotic, non-transformed host cells will die on the medium, while transformed host cells will survive. The plates are then incubated for a predetermined time to allow for the colonization of transformed host cells, and to induce cell samples from these plates for inducers to induce expression and production of peptides contained in the insert plasmid. Transfer to another plate containing further. If transformed host cells continue to grow in this environment and form normal colonies, it can be inferred that the expressed and produced peptides do not kill and / or inhibit the host cells. On the other hand, if the host cells do not form any colonies or the colonies are reduced compared to normal growth, it is clear that the derived peptides have an antibacterial effect on the host cells. A description of the screening strategy for conventional agarose plates is
Given in FIG. However, plate assays involve time consuming and lengthy procedures, and in a further preferred embodiment said screening or separation step is performed in a microtiter assay as described in the art.
In this type of assay, the liquid host cell culture is arranged in each well of a microtiter plate with a single or only a few cells containing different inserted nucleotide sequences, or otherwise only one examined. Alternatively, several nucleotide sequences are guaranteed to be contained in each well (eg multiple host cells containing the same inserted nucleotide sequence). The host cells in each well can be cultivated by the addition of nutrient medium, and copying or replication of the microtiter plate described above,
It can be prepared by moving the sub-samples to an additional test plate. A medium containing an inducer is then added to each well, and the growth of the host cell culture in each well in culture is observed, for example by measuring light diffraction through a suspension of the cells in each well. . If the growth of the host cell was not affected by the expressed peptide,
The number of cells in this well will normally increase, and the light diffraction of the cell suspension as measured through the well will increase. However, if the growth of the host cells was affected by the expressed peptide, the cell number in this well would be
There will be a decrease compared to normal growth, and the change in light diffraction of the suspension of cells will also be small. A description of the screening strategy for microtiter plates is given in Figure 3. In a third, and most preferred embodiment, the screening or separating step is performed, for example, by Gant VA, Warnes G., Phillips I.
. and Savidge GF; "The application of flow cytometry to the study of b
acterial responses to antibiotics "; J. Med. Microbiol .; 1993; 39; pp.147
-154 to preparative fluorescence assisted cell sorting according (F luorescence A ssiste
d C ell S ortiong) (it may be carried out by utilizing a FACS) device. Devices of this type are extensively described in the art, for example by the manufacturers of the above devices. For use in this approach, a live / dead discrimination probe (eg, a fluorescent or colorimetric probe), which is an indicator of proliferation of each cell, is incorporated into the host cell. Suitably, the inducer and the life-and-death discrimination probe are added to a liquid culture of exponentially growing host cells, and killed or growth-inhibited microorganisms are identified and recovered.

By holding the integrated probe, it is possible to observe the life or death of the cell by measuring the fluorescence in the cell by exposing the cell to excitation light having a wavelength suitable for the probe, for example, If the fluorescence of the probe is measured, the cell is alive or vice versa. With the FACS machine, cells exhibiting the required characteristics can be selected with considerable speed and accuracy, and fluorescence measurements can also be aided by the fact that they are sensitive. In the method of the present invention, the FAC
The S device comprises the screening or selection step (c) of selecting live cells and transformed host cells and / or the selection step of selecting killed and / or inhibited cells after expression of the peptide (e). ) Or can be combined with the plate and / or microtiter plate technology described above. Many suitable fluorescent probes for this purpose are commercially available, for example from Molecular Probes, Eugene, Oregon, USA. In the use of said probe, whether it damages or degrades the membrane structure, reduces or eliminates its ability to penetrate the membrane, or allows the particular probe to interact with intracellular targets (eg DNA). Please consider. Examples of said probes are intended, without limitation, to permeate dead and / or damaged cells and to fluoresce the nucleic acids of the cells (eg SYTOX ™ green nucleic acid stain) or live cells. A molecule that is a probe (eg, SYTO ™ live cell nucleic acid stain) intended to transmit and fluoresce
Includes SYTO (X) ™ nucleic acid stain from Probes. Procedures for using the probe are available from its manufacturer. Fluorescent redox probes (which can be used for membrane permeability) can also be used by Rodriguez, GG, Phipps D., Ishigu.
ro K. and Ridgway HF; "Use of a Fluorescent Redox Probe for Direct Vis
ualization of Actively Respiring Bacteria ", Applied and Environmental Mi
Use the 5-quano-2,3-ditoluyltetrazolium chloride probe described in crobiology, 1992, pp.1801-1808, or Jepras RI, Paul FE, Pear.
son SC and Wilkinson MJ; "Rapid Assessment of Antibiotic Effects on
Escherichia coli by bis- (1,3-Dibutylbarbituric Acid) trimethine Oxonol a
nd Flow cytometry "; Antimicrobial Agents and Chemotherapy; 1997; pp.2001
-Use the DiBAC ₄ (3) probe available from Molecular Probes, Inc.

The FACS device or conventional chiller is a Virta M, Akerman KEO, Saviranta P
., Oker-Blom C. and Karp MT; "Real time measurement of cell permeabili
zation with low-molecular-weight membranolytic agents ", Journal of Antim
It can also be adapted for use in bioluminescence by the techniques described in icrobial Chemotherapy; 1995; 36; pp. 303-315. Moreover, the colorimeter is more preferably used in a non-fluorescent cell sorting device intended for colorimetric measurement, for example Roslev P.
and King GM; "Application of a Tetrazolium Salt with Water-Soluble For
mazan as an Indicator of viability in Respiring Bacteria "; Applied and E
nvironmental Microbiology, 1993, pp. 2891-2896; A description of the screening strategy for FACS is given in Figure 1.

When applying the method of the invention to the screening of intracellular bioactive peptides, alternative preferred screening or selection strategies may be utilized, especially in eukaryotic cells, eg mammalian cells. Upon induced expression of the inserted nucleotide sequence, if it is bioactive against the host cell, the resulting peptide is
Cells that tear and / or degrade the cell membrane and are killed and / or affected cells are separated from unaffected cells by centrifugation or filtration. When centrifuging, the unaffected live cells may precipitate, while the nucleic acid material from the affected cells may be separated into the supernatant. This separation can also be done by filtering out the unaffected live cells. From this separation step amplification and mutations can be performed as described above.

Once the optimal peptide has been identified, the sensitivity of the peptide to salt concentration, ionic strength, pH and / or sensitivity of normal mammalian cells to the peptide, in particular, is determined by its intended use. Other tests can be done where the dependent test is done.

Uses of Antimicrobial Peptides The antimicrobial peptides discovered by the methods of the present invention can be utilized in many fields of application. One of the preferred fields is as an alternative to chemical preservatives, for example in the preservation of food / feed, paint formulations, detergent formulations, cosmetics or other personal care products. The peptide can be used in a conservative medical device such as an artificial joint implant or an intravenous tube by coating the material with a coating containing the peptide. Said peptides disinfect and / or sterilize and / or sterilize microorganisms on the surface of interest, eg as disinfectants for the treatment of biofilms in the cleaning industry
Alternatively, it can be actively applied to inhibit. One of the preferred uses is for humans and / or
Alternatively, it is a peptide preparation for treating microbial infection and / or tumor on the body of an animal or on the surface of skin or mucous membrane. The use of the screening method of the invention is
To discover highly physiologically active peptides that can kill and / or inhibit microorganisms, but have little or no negative effect on normal mammalian and / or eukaryotic cells It is intended to be a universal tool.
The peptides may be formulated for oral administration, for intravenous or subcutaneous injection, or as an ointment.

Examples Example 1: E. coli by expression of various antimicrobial peptides. E. coli growth inhibition model AMP, namely CAP18, PR39, andropine, Bac5, B
ac7, Clavanin A, Clavanin AK (Clavanin A mutant), Stierin D
, And the stierin C gene were synthesized with oligo DNA in a standard PCR reaction and cloned in this SES to assess their ability to identify AMPs with improved bioactivity.

Plasmid Two experimental series were generated: the pHH series (using the plasmid pBAD / gIIIA) was derived from the E. coli strains where the peptide was allowed to interact with the cell membrane . It was possible to export the AMP to the periplasmic space of E. coli. In the pHHA series (using plasmid pHHA), the peptide lacked a signal sequence and the corresponding occurrence was retained in the cytoplasm.

One of the parent plasmids used, pBAD / gIIIA, is commercially available from Invitrogen. It is a tightly regulated E. It is an expression vector derived from pUC intended to express a recombinant protein in E. coli . This plasmid was prepared as described in E. coli with no expression of the recombinant peptide in the absence of inducer in the growth medium . It allows the cloning of peptides and proteins that are toxic to E. coli . However, transcription and peptide synthesis based on it
Can be widely induced.

Selection Since all AMPs first attack or penetrate the target organism from the outside, the SES
The success of is often required for the AMP to be exported out of the cell. In this system, the AMP is secreted into the periplasmic space from locations that allow it to interact with its cellular targets, such as components in the cell membrane, membrane or periplasmic space, or from sites that allow further diffusion through the outer membrane. Was done.

In the pHH series, the geneIII signal sequence in pBAD / gIIIA was placed in front of an inducible promoter to achieve secretion of the peptide / protein. GeneIII is one of the few capsid proteins from filamentous phage fd that encodes pIII. pIII is synthesized by an 18 amino acid N-terminal signal sequence and requires the bacterial secretory system for insertion into the membrane. The signal sequence is removed after passing through the inner membrane, thus leaving the original peptide. The Nco I restriction site immediately follows the split site in the signal sequence.

In the other parental plasmid series, pHHA, the peptide was synthesized without the signal sequence, and the corresponding one was retained in the cytoplasm. The pHHA plasmid had pBA only at the point where the geneIII was deleted.
Different from D / gIIIA. This deletion was created by the introduction of an additional Nco I site that overlaps the transcription start site. Therefore, the signal sequence was removed by restriction with NcoI and the plasmid religated to p
Created HHA.

In a replasmid system (pBAD / gIIIA and pHHA), the AMP
The gene is introduced as a Nco I- Xba I fragment.

Regulation of Inducible Promoter pBAD, which is an inducible promoter used, contains two proteins, AraC.
And cAMP-CRP regulates both positive and negative. In the presence of arabinose, expression from the promoter is initiated, whereas in the absence of arabinose, very low levels of expression occur from the promoter. Non-induced levels are even further suppressed by growth in the presence of glucose.
Glucose acts to reduce cAMP levels which reduces the binding of the cAMP-CRP to the promoter region of pBAD. By lowering the cAMP level,
Decrease transcription activity. This is ideal if the peptide of interest is very growth inhibitory or toxic to the host cell. In short, by altering the activity of the two modulators, the protein expression level can be optimally manipulated for potential toxicity or expression of the native gene.

[0065] The genes of the various AMP touching the myc epitope and 6x His tag destination C-terminus, and myc epitope within reading frame 6
Clone before the xHis tag. A TAG stop codon separated the AMP gene and the sequences encoding the two epitopes. This is a normal E. In E. coli , it means that only AMP is synthesized by the induction of transcription. The termination of translation at the TAG stop codon is determined by the fusion protein in E. When non-toxic to E. coli , it may be suppressed in various strains to allow the synthesis of AMP / myc / 6x His fusion proteins. This fusion protein is easily purified using the affinity of nickel (Ni ²⁺ ) resin and is easily detected using anti- Myc or anti-6x His antibodies. Following purification, cyanogen bromide decomposition
The two tags are separated from the AMP by digestion at a conveniently located methionine. This system allows for easy and convenient purification that allows confirmation using the conventional assay of the antimicrobial activity of the selected peptides.

The strain utilized in the host organism is E. E. coli TOP10 (commercially available from Invitrogen). It is ara BADC ⁻ and ara EFGH ⁺ .

Model AMPs below the AMP gene , namely CAP18, PR39, andropine, Bac
5, Bac7, clavanin A, clavanin AK (clavanine A mutant), stierin D, and stierin C active fragments were synthesized by standard PCR reactions using the oligo DNAs shown below. The AMP genes the PCR amplification was restricted with Nco I and Xba I, and ligated into the corresponding cloning sites in pBAD / gIIIA and PHHA. These ligation reaction mixtures were treated with chemically competent E. E. coli TOP10 and transformed into each clone. The AMP gene was finally confirmed by DNA sequencing.

Primers used for the synthesis of the AMP gene

[0069]

[Chemical 1]

[0070]

[Chemical 2]

[0071]

[Chemical 3]

[0072]   The amino acid sequence corresponding to the AMP gene is shown below.

[0073] lower case letters of the amino acid is not present in the natural AMP, closest cloning site (CCATGG, ATG codes for methionine) was created as a result of the cloning strategy to use the Nco I as. The natural codon usage of the gene was maintained.

[0074]

[Chemical 4]

[0075]

[Chemical 5]

E. coli due to expression of various AMPs. Growth inhibition of E. coli The following experiments were carried out by induction of endogenous AMP expression . It was performed to evaluate whether E. coli is growth-inhibited in liquid medium.

On the first day, the cells harboring the plasmids encoding the respective AMPs were
Seed in B + 100γ ampicillin and grown under non-inducing conditions at 37 ° C. with vigorous shaking. These overnight cultures were incubated on day 2 with fresh pre-warmed LB broth + 100γ ampicillin in the presence of different amounts of arabinose (0%, 0.001%, 0.01%, and 0.1%). Diluted 100-fold into. 100 μl of these freshly diluted cultures were transferred to microtiter plates and incubated at 37 ° C. with shaking. ELISA of the growth of the culture
It was measured at regular intervals at OD ₄₅₀ using a reader. The corresponding growth curves are shown in Figures 5-14.

Where the peptide is secreted into the periplasmic space (the parental plasmid pBAD / gI
In the pHH series (using IIA), expression of all AMPs except andropine significantly inhibited bacterial growth. E. coli with a similar genotype ( ara BADC ^- and ara EFGH ⁺ ) . Similar results were obtained when other strains of E. coli were used (data not shown). Andropine has been reported to require sufficient salt concentration to fold and exert its antibacterial activity; an observation that could explain the weaker inhibition seen. Non-growth inhibition is described in pII
It is evident in the strain carrying the pBAD / gIIIA control plasmid expressing 38 amino acids of the Myc / HIS6 control peptide fused to the I signal sequence. From this, it can be concluded that the expression itself of the peptide fused to the pIII signal sequence is E. coli . It can be determined that it does not significantly inhibit the growth of E. coli . The results are shown in Figures 5b-14b.

Different patterns of growth inhibition were observed in the pHHA series where the peptide was retained cytoplasmically. In this context only one subset of the AMPs exerted growth inhibition. This difference probably reflects the different mode of action of the AMP. It is suggested in the literature that peptides that do not depend on membrane permeability or peptides that have intracellular targets (eg, the proline-arginine rich peptide family Bac5, Bac7, and PR39) interact with and inactivate nucleic acids. ) Is expressed and retained in the cell and is expected to affect its viability. The results are shown in FIGS.
Shown in.

E. coli on solid medium due to expression of various AMPs. Growth inhibition of E. coli The following experiments were performed on E. coli by induction of endogenous AMP expression . It was carried out to evaluate whether E. coli was growth-inhibited on solid medium.

On day 1, cells harboring the plasmid encoding AMP were seeded in LB + 100γ ampicillin and grown at 37 ° C. under vigorous shaking under non-induced conditions. These overnight cultures were incubated on day 2 with fresh, pre-warmed LB broth +10.
L diluted 100-fold in 0γ ampicillin and containing 100γ ampicillin and different amounts of arabinose (0%, 0.001%, 0.01%, and 0.1%).
3 μl spots were spotted on a B agarose plate. These agarose plates were then incubated overnight at 37 ° C. The growth of bacterial clones was recorded the next day. Inhibition of proliferation was observed to demonstrate a correlation with the amount of arabinose present. The inhibition pattern reflected the results observed during growth in liquid medium.

Mutation Library To examine whether the suicide expression system is able to discriminate between peptide variants with different antibacterial activity, a mutation library of the 9 AMP genes was constructed using PCR and 0. It was prepared using 5 mM manganese chloride. For illustration purposes, a random excision of mutant Stierin C clones was selected and analyzed for inducer-dependent growth inhibition. Mutant AMPs have been identified by those that appear to have altered bioactivity (Figure 15). A subset of the mutants were sequenced, and all clones exhibiting altered bioactivity was confirmed that they are not equal with the w t (wild-type).

[Brief description of drawings]

FIG. 1 shows a diagram showing the flow of method steps in a SES system using a fluorescence-assisted cell sorting (FACS) device for the identification of modified antimicrobial peptides (AMPs), Where A is a mutant antimicrobial peptide (AMP) in a bacterial host cell
B is the removal of killed bacteria via FACS; C is the library of
Transcriptional induction; D is FACS-mediated selection of nonviable bacteria and E is PCR amplification, shuffling of amplified genes, cloning, and transformation. The following symbols were used :.

[Table 1]

FIG. 2. AM modified with conventional agarose plates using solid medium.
A diagram showing the flow of the screening strategy for the identification of P is shown, where A
Is the distribution of bacterial clones on agarose plates; B is the production of replica plates; C is the induction of transcription (development) and D is the characterization of colonies, eg characteristics, AMP sequences. , A colony of dead or inhibited cells, P
Includes CR amplification, gene shuffling and cloning.

FIG. 3. AM modified by microtiter plate with liquid medium.
Figure 2 shows a diagram depicting a flow of screening strategies for the identification of P, where A is the distribution of bacterial clones into microtiter wells; B is the replica
Plate generation; C is transcription induction and D is colony characterization, eg traits, AMP sequences, identification of dead or inhibited cell colonies, PCR amplification, gene shuffling and cloning. including.

FIG. 4 shows that E. coli transformed with DNA encoding AMP. E. coli, where the expression of AMP can be induced by an arabinose inducer. Levels of inducer are shown in the vertical direction. Testing different AMP's,
Where 1 is andropine; 2 is Bac7; 3 is Bac5; 4 is stierin D; 5 is stierin C; 6 is PR39; 7 is ClavA; 8 is ClavAK; 9 is CAP18
And 10 is pBAD. Different E. The effect on E. coli colonies can be confirmed visually.

Figure 5 A: E expression androsta pin (PHHA1000- androsta-pin) is induced. Growth curves at different levels of E. coli arabinose inducer, wherein the andropine is retained in the cytoplasm. Growth of cell suspension 450
Observed by OD measurement at nm. Inducer levels are given in% w / w. B: E. coli in which expression of andropine (PHH1000-andropine) is induced . Growth curves of E. coli arabinose inducers at different levels, wherein the andropine is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

FIG. 6A: E. coli in which expression of Bac7 (PHHA1100-Bac7) is induced . FIG. 3 is a growth curve of the arabinose inducer of col i at different levels, wherein B
ac7 is retained in the cytoplasm. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w. B: E. coli in which expression of Bac7 (PHH1100-Bac7) is induced . E. coli arabinose inducer growth curves at different levels, wherein the Ba
c7 is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

FIG. 7: A. E. coli in which expression of Bac5 (PHHA1200-Bac5) is induced . FIG. 3 is a growth curve of the arabinose inducer of col i at different levels, wherein B
ac5 is retained in the cytoplasm. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w. B: E. coli in which expression of Bac5 (PHH1200-Bac5) is induced . E. coli arabinose inducer growth curves at different levels, wherein the Ba
c5 is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

8 A: Suchierin D E expression of (PHHA1300- Suchierin D) is derived. Growth curves of different levels of E. coli arabinose inducer at different levels, wherein the stierin D is retained in the cytoplasm. Growth of cell suspension 450
Observed by OD measurement at nm. Inducer levels are given in% w / w. B: E. coli in which the expression of stierin D (PHH1300-stierin D) is induced . Growth curves of E. coli arabinose inducers at different levels, wherein the stielin D is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

9 A: Suchierin C E expression of (PHHA1400- Suchierin C) is induced. Growth curves of different levels of E. coli arabinose inducer at different levels, wherein the stierin C is retained in the cytoplasm. Growth of cell suspension 450
Observed by OD measurement at nm. Inducer levels are given in% w / w. B: E. coli in which the expression of stierin C (PHH1400-stierin C) is induced . Growth curves of different levels of E. coli arabinose inducer at different levels, wherein the stierin C is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

FIG. 10 A: E. coli in which expression of PR39 (PHHA1500-PR39) is induced . FIG. 3 shows the growth curves of arabinose inducers of col i at different levels, where P
R39 is retained in the cytoplasm. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w. B. E. coli in which expression of PR39 (PHH1500-PR39) is induced . E. coli arabinose inducer growth curves at different levels, wherein PR
39 is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

[11] A: Kurabanin A E expression of (PHHA1600- Kurabanin A) is derived. Growth curves of E. coli arabinose inducers at different levels, wherein the Clavanin A is retained in the cytoplasm. Growth of cell suspension 450
Observed by OD measurement at nm. Inducer levels are given in% w / w. B: Expression of Clavanin A (PHH1600-Clavanin A) is induced in E. coli . Growth curves of E. coli arabinose inducers at different levels, wherein the Clavanin A is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

[Fig. 12]   A: Expression of clavanin AK (PHHA1700-clavanine AK) was induced
RuE. coliGrowth curves of different levels of arabinose inducer of
Here, the clavanin AK is retained in the cytoplasm. Growth of cell suspension
Observed by OD measurement at 450 nm. Inducer levels are given in% w / w
It   B: Induction of expression of clavanin AK (PHH1700-clavanine AK) E. coli Growth curves of different levels of arabinose inducer of
The clavanin AK is then secreted into the periplasmic space. Proliferation of 45 of cell suspension
Observed by OD measurement at 0 nm. Inducer levels are given in% w / w.

FIG. 13A: E. coli in which expression of CAP18 (PHHA1800-CAP18) is induced . A growth curve for different levels of arabinose inducer of c oli, wherein said CAP18 is retained in the cytoplasm. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w. B: Expression of CAP18 (PHH1800-CAP18) is induced in E. coli . A growth curve at different levels of arabinose inducer of co li, wherein said CAP18 is secreted to the periplasmic space. Proliferation of OD of cell suspension at 450 nm
It was observed by measurement. Inducer levels are given in% w / w.

FIG. 14A: E. coli in which expression of the control peptide Myc / HIS6 is induced . growth curves of E. coli arabinose inducers at different levels, wherein the Myc / HI
S6 is retained in the cytoplasm. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w. B: Expression of control peptide Myc / HIS6 is induced in E. growth curves of E. coli arabinose inducers at different levels, wherein the Myc / HI
S6 is secreted into the periplasmic space. Proliferation was monitored by OD measurement of the cell suspension at 450 nm. Inducer levels are given in% w / w.

FIG. 15   Induction of expression of Stierin C mutants from randomly picked mutants E. coli Is a growth curve of. Randomly sampled black of a Stierin C mutant
The zones are numbered from # 1 to # 10. Inducer levels given in% w / w
To be

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] July 9, 2001 (2001.7.9)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 31/04 171 A61K 37/02 35/00 37/48 (81) Designated country EP (AT, BE, CH) , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN , GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW

Claims (18)

[Claims] 1. A method for sequencing a pool of nucleotide sequences for selecting a nucleotide sequence encoding a peptide, comprising: a) an enzyme having less than 100 amino acid residues optionally linked to a signal peptide, or A plasmid is ligated to the pool of nucleotide sequences operably linked to an inducible promoter so as to express the peptide which is a mature peptide, and b) a host cell sensitive to the peptide is bound by the bound plasmid. Transforming, c) screening the transformed host cells to select live cells, and d) the expression of the nucleotide sequence in the presence of an inducer to induce expression of the nucleotide sequence. Culturing the cells, e) selecting the cells according to the effect of the above inducers on cell proliferation, It said method from to f) above selected cells to recover a nucleotide sequence encoding the peptide, comprising the. 2. The method of claim 1, wherein the pool of nucleotide sequences is generated by random mutagenesis, gene shuffling, or degenerate gene synthesis. 3. The method according to claim 1, wherein the peptide is an enzyme. 4. The method according to claim 1 or 2, wherein the peptide is a short peptide consisting of less than 100 amino acid residues, preferably an antibacterial peptide or an antitumor peptide. 5. The above-mentioned peptide is an antibacterial peptide or antibacterial active against bacteria.
And the host cell is a bacterial cell (especially Escherichia coli ( E. coli ) Or Bacillus (Bacillus)), Filamentous fungi (especially asperg
Gillus (Aspergillus)) Or yeast cells (especially Candida (Can dida ) Or Saccharomyces (Saccharomyces))
The method of claim 4. 6. The method according to claim 4, wherein the peptide is an antitumor peptide and the host cell is a mammalian cell, in particular a tumor cell. 7. The peptide is a short peptide of less than 100 amino acid residues, and the bond provides the peptide without extension or with an extension of the N-terminal 1 to 5 amino acids, and optionally a signal. -The method according to any one of claims 4 to 6, which is for expressing a peptide. 8. The method according to claim 1, wherein the selection of steps c) and e) is performed using an agarose plate. 9. The method according to claim 1, wherein the selection of steps c) and e) is carried out in microtiter wells. 10. The method according to claim 1, wherein steps c) and e) are selected using a FACS machine. 11. The method according to claim 1, wherein the selection of steps c) and e) is carried out by centrifugation or filtration. 12. Use of the method according to any one of claims 1 to 11 for the discovery and manufacture of formulations for the treatment of the human or animal body. 13. In vitro homologous DNA, wherein said gene shuffling comprises cleaving a homologous template double-stranded polynucleotide into random fragments, followed by homologous reassociation of said fragments with a full-length gene. The method of claim 2 including shuffling. 14. The gene shuffling comprises formation of a library of recombined homologous polynucleotides constructed from an input DNA template and random DNA primers by template shift induced during in vitro DNA synthesis. The method described in 2. 15. The method of claim 14, wherein the random DNA primers are utilized to randomly initiate DNA synthesis on the mutant DNA template to be combined. 16. The method of claim 2, wherein the gene shuffling comprises the use of primers with less than 30 base pairs that harbor the mutation to be merged. 17. The gene shuffling encodes the entire gene 1
The method according to claim 2, which comprises the synthesis of the above degenerate DNA primer. 18. The gene sequence diversity comprises as a starting material a library of cDNA or a library of randomly generated whole genomic DNA fragments.
The method of claim 1.