JP2002537758A - Shuffling of codon-modified genes - Google Patents

Abstract

  (57) [Summary] Methods for recombination of a library of codon-altered nucleic acids are provided. The nucleic acid may contain conservative or non-conservative alterations in the coding sequence in addition to the codon changes as compared to the wild-type sequence. In addition to methods for making novel proteins, methods for generating vectors with reduced rates of reversion to wild-type and attenuated viruses are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application was filed on September 29, 1998 by Patten and Stem.
USSN 60/10, attorney registration no.
2,362 "SHUFFLING OF CODON ALTERED GEN
ES "and Patten and Ste, filed January 29, 1999.
USSN 60/1, attorney registration no.
17,729 "SHUFFLING OF CODON ALTERED GE
NES is not a provisional application. This application is also related to USSN6, filed February 5, 1999, by Crameri et al., Agent Registration No. 02-296.
0 / 118,813 "OLIGONUCLEOTIDE MEDIATED N
UCLEIC ACID RECOMBINATION "; and January 1999
Et al., Agent Registration No. 02-296-1, filed on March 24.
, USSN 60 / 141,049 "OLIGONUCLEOTIDE
MEDIATED NUCLEC ACID RECOMBINATION "
is connected with.

BACKGROUND [0002] The genetic code is highly degenerate. All DNAs encoding amino acids /
RNA triplets (codons) are typically, except for ATG / AUG (encoding methionine) and TGG / UGG (encoding tryptophan),
Without altering the sequence of the protein encoded by the corresponding nucleic acid sequence,
Can be changed. Approximately, on average (change in distribution of amino acids between proteins), each code triplet can be replaced in approximately three different ways. Because there are 61 codons encoding 20 amino acids (for all 64 codons encoding 20 amino acids, there are 3 additional triplets encoding stop codons). This indicates a possible sequence diversity of approximately 3 ⁿ possible sequences encoding a given protein, where n is the length of the protein in amino acids. As can be easily found, even for moderately long proteins, the number of potential nucleic acids, which can encode proteins, exceeds the number of physical particles in the universe (about 10 Estimated to be ⁸⁰ particles).

[0003] This tremendously large potential sequence space for individual proteins has interesting evolutionary relationships. For example, hypermutable viruses (eg, HI
V and other retroviruses) typically accumulate non-random mutations based in part on the particular codons used to encode recognition molecules (eg, in the viral envelope proteins). Go one step further in the host immune system. Mutations are not random. This is because viruses are selected for their ability to mutate into a form that is not quickly recognized by the host immune system. As a result, the virus is selected, for example, to have a non-random set of codons encoding the envelope protein, whereby
For example, by making specific point mutations that create specific alterations in the protein structure, the virus will rapidly shift form.

[0004] Codon usage is also not random within a species. All possible t-RNA
By preferentially producing a subset of the cells, cells can conserve energy and optimize or even regulate the efficiency of a cellular translation system.
This fact has long been empirically recognized and often allows researchers to first easily determine the reading frame of a given nucleic acid sequence by simply considering the codons caused by the potentially different reading frames. Make it possible. One consequence of this "species codon bias" is that the proteins in the species have a restricted set of possible mutations that can occur, for example, as a result of point mutations. This limits the possible rate of evolution of the protein.

[0005] In addition to the diversity of the coding sequences of nucleic acids encoding any given protein, it is now evident that protein sequences themselves are extremely degenerate.
Often, many of the amino acid residues that make up a protein can be replaced with structurally similar amino acid units without significantly altering the tertiary structure of the protein.
Thus, determining which residues alter or improve the desired properties of the protein can be difficult.

For commercially valuable proteins, it is desirable to be able to utilize a mutated spectrum that is different from that of the native protein. The present invention provides this and many other features, which will be apparent based on the following complete description.

SUMMARY OF THE INVENTION [0007] The present invention provides a method of accessing a completely different mutated spectrum for a selected protein than is available in naturally occurring nucleic acids encoding the protein. This increases the type and rate of forced evolution for the selected protein, thereby allowing for rapid improvement of any detectable characteristics of the protein. In this method, the nucleic acid is synthesized using altered codon usage and / or the nucleic acid encodes one or several amino acid residue changes when compared to the selected protein ( Here, amino acid changes and codon usage changes may be conservative or non-conservative). The resulting codon / amino acid modified nucleic acid (s) may be used with DNA shuffling techniques (typically by an iterative shuffling method) either with natural nucleic acids or with each other (or both). Recombinant). The nucleic acid or encoded protein is then screened for the desired properties.

Accordingly, the present invention provides a method for producing a codon-altered nucleic acid. In this method, a first nucleic acid encoding a first polypeptide sequence is selected.
A plurality of codon-altered nucleic acid sequences, each of which encodes a first polypeptide or an altered form thereof, are then selected (eg, a library of codon-altered nucleic acids is a component of the library). Or in a biological assay recognizing activity), and the plurality of codon-altered nucleic acid sequences are recombined to yield a target codon-altered nucleic acid encoding a second protein. The target codon-altered nucleic acid is then optionally screened for a detectable functional or structural property, including comparison to a property of the first polypeptide. The purpose of such a screen is to identify polypeptides that have structural or functional properties that are equivalent to or better than the first polypeptide. Nucleic acids encoding such polypeptides can be introduced into a cell, vector, virus, attenuated virus (eg, as a component of a vaccine or immunogenic composition), transgenic organism, etc., into a target codon-modified nucleic acid. It can be used in essentially any desired procedure, including:

[0009] Kits and compositions for performing the method are also provided. These include one or more of a recombinant mixture of cells and a substrate (eg, a nucleic acid with altered codon usage), a container, instructions for performing the method, and the like.

Definitions Unless clearly indicated to the contrary, the following definitions supplement the definitions of terms known in the art.

[0011] As used herein, a "recombinant" nucleic acid is produced recombinantly between two or more nucleic acids, or any nucleic acid made by an in vitro or artificial process. , Nucleic acids. The term "recombinant," when used in reference to a cell, indicates that the cell contains (and optionally replicates) a heterologous nucleic acid or expresses a peptide or protein encoded by the heterologous nucleic acid.
Recombinant cells can contain genes that are not found in the cell's native (non-recombinant) wild-type form. Recombinant cells can also contain genes that are found in the cell's natural form when the genes are modified and reintroduced into the cell by artificial means. The term also includes cells containing nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell; such modifications include gene substitutions, site-specific mutations, chimeras And those obtained by related techniques.

A “codon-altered” nucleic acid is a first nucleic acid that encodes a first polypeptide that is similar to or identical to a naturally-occurring polypeptide encoded by a naturally-occurring nucleic acid. It is. Here, the first nucleic acid utilizes a plurality of codons encoding the first polypeptide, which is different from the codon of a naturally occurring nucleic acid encoding a naturally occurring polypeptide.

“Nucleic acid sequence” refers to a nucleic acid (eg, RNA, DNA, or a modified, isolated, recombinant, or natural form thereof) or the primary structure (sequence) of a nucleic acid. Refers to any representation of a nucleic acid, such as a sequence of letters.

“Polypeptide sequence” is a character sequence that indicates a polypeptide (altered, isolated, recombinant, or natural form thereof) or the primary structure (amino acid sequence) of a polypeptide. Or other representations of the polypeptide, such as character string information.

A “modified form” of a reference polypeptide is a target polypeptide having a sequence that is similar, but not identical, to the reference polypeptide. The sequence of the target polypeptide can differ from the reference polypeptide by conservative or non-conservative substitutions of the reference polypeptide sequence. As described in more detail above, different nucleic acids encoding different target polypeptides having different non-conservative substitutions as compared to the reference polypeptide encode a more similar target polypeptide to the reference polypeptide It can be recombined to produce a recombinant nucleic acid.

The “plural forms” of a selected nucleic acid refer to multiple homologs of the nucleic acid. The homolog may be derived from a naturally occurring homolog (eg, two or more homologous genes or derivatives thereof) or may be due to the artificial synthesis of one or more nucleic acids having related sequences. , Or by altering one or more nucleic acids to give rise to related nucleic acids. Nucleic acids are homologous when they are derived, naturally or artificially, from a common ancestral sequence. During natural evolution, this occurs when more than one progeny sequence results from the parent sequence over time, ie, due to mutation and natural selection. Under artificial conditions, branching occurs, for example, in one of two ways. First, a given sequence can be artificially recombined with another sequence (eg, as occurs during a representative cloning) to produce progeny nucleic acids. Alternatively, the nucleic acid can be newly synthesized by synthesizing a nucleic acid having a sequence different from that of a predetermined parent nucleic acid sequence.

In the absence of apparent knowledge of the ancestry of two nucleic acids, homology is typically inferred by sequence comparison between the two sequences. If two nucleic acid sequences show sequence similarity, the two nucleic acids are presumed to share a common ancestor. The exact level of sequence similarity required to establish homology will vary in the art depending on various factors. For the purposes of this disclosure, two sequences are
If they share sufficient sequence identity to allow recombination to occur between two nucleic acid molecules, or if codon changes that result in two or more nucleic acids capable of performing recombination can be made , Are considered homologous. Typically, nucleic acids require closely similar regions, located approximately the same distance apart, to allow recombination to occur.

The terms “identical” or percent “identity” in the context of two or more nucleic acid or polypeptide sequences refer to the sequence comparison algorithms described below (or other algorithms available to those of skill in the art). A specific percentage of amino acid residues or nucleotides that are identical or identical when measured using one or by visual inspection, compared for greatest correspondence, and aligned Refers to two or more sequences or subsequences having

The phrase “substantially identical” in the context of two nucleic acids or polypeptides refers to the greatest correspondence when using one of the following sequence comparison algorithms or as determined by visual inspection: When compared and aligned,
At least about 40%, 50%, 60%, or preferably about 70%, or 8
Refers to two or more sequences or subsequences that have 0% or more, or most preferably, 90-95% nucleotide or amino acid residue identity. Such "substantially identical" sequences are typically considered to be homologous.
Preferably, "substantial identity" exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably over a region of at least about 100 residues. Are substantially identical over at least about 150 residues or the entire length of the two sequences being compared.

For sequence comparison and homology determination, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, sequence coordination is designed (if necessary), and sequence algorithm program parameters are designed. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) compared to the reference sequence, based on the designed program parameters.

The optimal alignment of sequences for comparison is, for example, Smith and Wat
erman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needlmen and Wunsch, J. Am. Mo
l. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad.
Sci. USA 85: 2444 (1988), by the computerized implementation of these algorithms (Wisconsin).
Genetics Software Package, Genetics
Computer Group, 575, Science Dr. , Madis
on, WI, GAP, BESTFIT, FASTA, and TFASTA), or by visual inspection (see generally Ausubel et al., supra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm. This is Altschul et al.
J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analysis is available from National Cen.
ter for Biotechnology Information (ht
tp: // www. ncbi. nlm. nih. Gov /) is publicly available. The algorithm involves first identifying a high-scoring sequence pair (HSP) by identifying short words of length W in the interrogation sequence. It either matches or satisfies some positive value threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score limit (Altschul et al., Supra).
These first neighbor word hits act as seeds for initiating searches to find longer HSPs containing them. This word hit is then expanded in both directions along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for matching residue pairs; always> 0) and N (penalty score for mismatching residues; always <0). . For amino acid sequences, a score matrix is used to calculate the cumulative score.
The expansion of word hits in each direction stops when: the cumulative alignment score drops by a quantity X from its maximum achieved; the cumulative score is:
Zero or less due to accumulation of one or more negative score residue alignments; or when reaching the end of either sequence. The parameters of the BLAST algorithm, W, T, and X, determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences)
1 word length (W), 10 expected value (E), 100 cutoff, M = 5, N =
-4, and a comparison of both strands is used as default. For amino acid sequences, the BLASTP program gives a wordlength (W) of 3, an expected value (E) of 10,
And the BLOSUM62 score matrix as default (He
nikoff and Henikoff (1989) Proc. Natl. Aca
d. Sci. ScL USA 89: 10915).

[0023] In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (eg, Karlin and Altschul (1993) Proc. Natl. Acad. .Sci.U
SA 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)). This provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, if the minimum total probability of comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is: It is considered similar to the reference sequence.

Another indication that two nucleic acid sequences are substantially identical / homologous is that the two molecules hybridize to each other under stringent conditions. The phrase “specifically hybridizes” includes where the sequences are present in complex mixed (eg, whole cell) DNA or RNA, and includes the ability of a molecule to bind to only a particular nucleotide sequence under stringent conditions. Refers to binding, duplex formation, or hybridization. "Substantially bind" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and reduces the stringency of the hybridization medium to achieve the desired detection of the target polynucleotide sequence. Including minor incompatibilities that can be achieved by

In the context of nucleic acid hybridization experiments (eg, Southern and Northern hybridizations), “stringent hybridization conditions and“ stringent hybridization wash conditions ”are sequence-dependent and have different environmental parameters. Different below. The longer sequence (s) with a higher G: C content will still hybridize at higher temperatures (or lower salt concentrations). Extensive guidance on nucleic acid hybridization can be found in Tijssen (1993) Laboratory Tec.
hniques in Biochemistry and Molecula
r Biology--Hybridization with Nuclei
c Acid Probes part I chapter 2 "Overv
view of principals of hybridization a
nd the strategy of nucleic acid prob
eassays "found in Elsvier, New York.

In general, high stringency hybridization conditions and washing
Conditions are defined as the melting temperature (T.sub.T) for a particular sequence at a defined ionic strength and pH. _m ) Is selected to be about 5 ° C. lower. Typically, "stringent
Under `` normal conditions, '' the probe will hybridize to its target subsequence, but
It does not hybridize to relevant (non-homologous) sequences.

T _m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Extremely stringent conditions are chosen to be equal to _Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than 100 complementary residues on a filter in a Southern or Northern blot is at 42 ° C containing 50 mg of heparin containing 1 mg of heparin.
% Formamide and hybridization is performed overnight. An example of high stringency washing conditions is 0.15 M NaCl at 72 ° C. for about 15 minutes. An example of stringent wash conditions is 0.2 × SSC at 65 ° C. for 1 hour.
5 minute wash (Sambrook, supra, for SSC buffer description)
. Often, high stringency washes are preceded by low stringency washes to remove background probe signals. For example, an exemplary moderate stringency wash for duplexes of more than 100 nucleotides is 1 × SSC at 45 ° C. for 15 minutes. For example, 1
An exemplary low stringency wash for duplexes of greater than 00 nucleotides is 4-6 × SSC at 40 ° C. for 15 minutes. For short probes (eg, about 10-50 nucleotides), stringent conditions are typically less than 1.0 M Na ion (typically about 0.01 to 1
. It contains a salt concentration of 0 M Na ion concentration (or other salt), pH 7.0 to 8.3) and the temperature is typically at least about 40 ° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Generally, a signal to noise ratio of 2x (or greater), as observed for an irrelevant probe in a particular hybridization assay, indicates the detection of specific hybridization. If the signal to noise ratio is less than 2 × that of an unrelated probe (eg, a nucleic acid encoding a non-homologous protein), the nucleic acid in question will not hybridize under stringent conditions. Similarly, if the signal to noise ratio is less than 25% of that observed for a perfectly matched probe under stringent conditions,
Nucleic acids do not "hybridize under stringent conditions," as the term is used herein. This does not apply for highly stringent conditions. This is because stringency can be increased theoretically until only perfectly matched probes hybridize.

In one exemplary hybridization procedure, the target nucleic acid to be probed is blotted on a filter by any conventional method. Irrelevant nucleic acids (eg, plasmid vectors) (assuming the target nucleic acid has no homology to the target nucleic acid) are also blotted on the filter in approximately equal amounts. The filter is probed with a labeled probe complementary to the target nucleic acid. The experiment is repeated with increasing stringency of hybridization and washing conditions until the signal from hybridization of the labeled probe to a complementary target is 10-100 fold higher than to an irrelevant plasmid vector nucleic acid. Once these conditions are determined, as described above, the test nucleic acid is probed under the same conditions as the target. The signal from the labeled probe is 25% of the signal due to the binding of the probe to the target
To the extent or higher, the test nucleic acid "hybridizes under stringent conditions" to the probe. If the signal is less than 25%,
The test nucleic acid does not hybridize under stringent conditions to the probe.

Nucleic acids that do not hybridize to each other under stringent conditions are still recognizable as variant forms of the nucleic acid when the polypeptides they encode are substantially identical. This occurs, for example, such that a copy of the nucleic acid is made using the maximum codon degeneracy permitted by the genetic code. Such nucleic acids are not functionally equivalent due to differences in mRNA folding, changes in regulatory sequences, and the like, as described in detail herein.

Another indication that two nucleic acid sequences or polypeptides are in a variant form is that when tested with a polyclonal antiserum raised against the first polypeptide,
That is, a polypeptide encoded by a first nucleic acid immunologically cross-reacts with a polypeptide encoded by a second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.

A “conservatively modified variation” of a particular polynucleotide sequence is a polynucleotide variation that encodes the same or essentially the same amino acid sequence, or when the polynucleotide does not encode an amino acid sequence. Are polynucleotide variations that encode essentially the same sequence.
Due to the degeneracy of the genetic code, a number of functionally identical nucleic acids encode any given polypeptide. For example, codons CGU, CGC, CGA, CGG
, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon may be changed for any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations." These are a kind of “conservatively modified variations”.
Every polynucleotide sequence described herein which encodes a polypeptide also describes, as appropriate, all possible silent variations. This excludes cases where otherwise stated. One skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is usually the codon for methionine, and TGG, which is usually the codon for tryptophan), can be modified to yield peptides that are structurally identical.

In addition, one skilled in the art will recognize that a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in the encoded sequence may be altered, added, or deleted. Recognizing a "conservatively modified variation" is where the individual substitutions, deletions, or additions made, when the alteration results in the replacement of the amino acid with a chemically similar amino acid. A table of conservative substitutions that provide functionally similar amino acids is
It is well known in the art. Each of the following five groups contains amino acids that are conservative substitutions for each other: Aliphatic: glycine (G), alanine (A), valine (V), leucine (L)
, Isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); containing sulfur: methionine (M), cysteine (C)
Basic: arginine (R), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)
. Creighton (1984) Proteins, W.C. H. Freeman
See also and Company. In addition, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are also "conservatively modified variations."
It is. Sequences that differ by conservative variations are generally homologous.

When the term “isolated” is applied to a nucleic acid or protein, the nucleic acid or protein is bound to other cellular or other components with which it is naturally associated ( (E.g., components of a library).

The term “nucleic acid” refers to its deoxyribonucleotides or ribonucleotides and their polymers, in either single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids that have similar binding properties as a reference nucleic acid, and that include known analogs of naturally occurring nucleotides that are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also specifically includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. In particular, substitution of degenerate codons creates a sequence in which the third position of one or more selected (or all) codons has been replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., (1991) Nucleic Ac
id Res. 19: 5081; Ohtsuka et al., (1985) J. Am. Biol
. Chem. 260: 2605-2608; Cassol et al. (1992); Ro
(1994) Mol. Cell. Probes 8: 91-9
8). The term nucleic acid is generic to the terms "gene", "DNA", "cDNA", "oligonucleotide", "RNA", "mRNA" and the like.

“Nucleic acid derived from a gene” refers to a nucleic acid that has ultimately acted as a template for the synthesis of a gene or a subsequence thereof. Accordingly, mRNA, cDNA reverse transcribed from mRNA, RNA transcribed from cDNA, DNA amplified from cDNA
The RNA, etc., transcribed from the amplified DNA are all derived from genes, and the detection of such derived products is an indication of the presence and / or amount of the original gene and / or gene transcript in the sample. It is.

A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence.

A “recombinant expression cassette” or simply “expression cassette” is a recombinant or synthetic, nucleic acid element that can affect the expression of a structural gene in a host that is compatible with such a sequence. The nucleic acid construct produced by The expression cassette contains at least a promoter and, optionally, a transcription termination signal. Typically, a recombinant expression cassette includes a nucleic acid to be transcribed (eg, a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors that are essential or aid in affecting expression can also be used as described herein. For example, the expression cassette also includes a nucleotide sequence that encodes a signal sequence that directs secretion of the expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that affect gene expression, can also be included in the expression cassette.

DETAILED DESCRIPTION OF THE INVENTION In the present invention, the sequence diversity of the substrate for the DNA shuffling procedure is determined by using a codon-modified nucleic acid as a template and / or by selecting a selected wild-type protein. By using a template that encodes a protein having conservative or non-conservative amino acid modifications compared to

These codon-altered nucleic acids can be chemically synthesized (eg, standard artificial synthesis protocols (eg, those typically used by commercial sources from which nucleic acids can be ordered). Or using any of the various methods herein available to those skilled in the art. For example, using standard synthetic methods followed by polymerase and / or ligase mediated oligonucleotide ligation / recombination protocols, the oligonucleotide fragment corresponding to the desired codon-altered nucleic acid is converted to a full-length oligonucleotide fragment. Can be made to produce a nucleic acid.

The combination of codon usage alterations and coding alterations is sufficient to reduce hybridization or even eliminate under stringent conditions the codon-altered nucleic acid to the nucleic acid that naturally encodes the selected protein. Can be extensive. This dramatically alters the mutations caused by possible single nucleotide mutations, thereby providing access to great diversity for DNA shuffling protocols.

In addition, recombination and selection of such nucleic acids during a DNA shuffling procedure not only results in access to a different set of potential mutations, but also results in altered regulation of transcription or translation. Altered morphology, nucleic acid localization, mRNA stability, etc. can also occur. In addition, the altered hybridization properties of the codon-altered nucleic acids lead to an alteration in the ability of the nucleic acid to hybridize with potential recombination partners, which results in a diversity of available recombination during shuffling. Gender changes and eventually increases.

Further, “family shuffling” using codon-altered substrates,
It further increases the potential sequence diversity of the starting material for recombination. As currently practiced, family shuffling methods involve shuffling nucleic acids encoding sequence variants (eg, species or allelic homologs) of a given protein. In this method, the procedure is modified by creating a codon-altered version of the sequence variant to access additional molecular diversity during recombination. Additional diversity is achieved by conservatively and non-conservatively modifying the starting nucleic acid to encode non-naturally occurring sequence variants. Family shuffling can still be performed using homologs of relatively low identity. In such cases, the codons can be altered in one or more of the members of the family to increase the level of identity between the members, thereby recombining them using the methods of the invention. Increase the ability of

Gene shuffling and family shuffling improve and function the function of a protein (steps the type of reaction, the substrate or activity of a selected protein such as an enzyme, or the regulation or structure of the expressed component). Provide two powerful methods that can be used. In family shuffling, homologous sequences (eg, derived from different species, chromosomal locations, or due to synthetic alterations) are recombined. In gene shuffling, the signal sequence is mutated or otherwise altered and then recombined.

Generation and screening of high quality shuffled libraries
Provides DNA shuffling (or "directed evolution"). The availability of suitable high-throughput analytical chemistry to screen libraries allows for integrated high-throughput shuffling and screening of libraries to achieve the desired activity.

In one significant embodiment, oligonucleotides for constructing codon-modified nucleic acids are designed computationally (“in silico”). Recombinant nucleic acids with altered putative codons have also been described in silico, ie, essentially on February 5, 1999, in USSN 60/118854, Serifonov and Stemmer "METHODS FOR MAK."
ING CHARACTER STRINGS, POLYNUCLEOTIDE
S & POLYPEPTIDES HAVING DESIRED CHAR
ACTERISTICS ".

Furthermore, rather than producing codon-modified nucleic acids as substrates for recombination, families of nucleic acids can be recombined simply by appropriate selection of the relevant oligonucleotides. This is USSN 60/118, February 5, 1999.
, 813, submitted by Crameri et al., "OLIGONUCLEOTIDE."
MEDIATED NUCLEIC ACID RECOMBINATION
And Crameri et al., "OLIGONUCLEOTIDE MEDIATED, filed June 24, 1999 with USSN 60 / 141,049.
As taught in NUCLEC ACID RECOMBINATION, in combination with shuffling methods mediated by a family of oligonucleotides, in a gene reconstitution method for making recombinant nucleic acids,
That is, it is used by using codon-modified nucleic acid oligonucleotides as discussed herein. This technique can be used to recombine homologous or even heterologous nucleic acid sequences. In the context of the present invention, oligonucleotides corresponding to a family of codon-modified nucleic acids are shuffled.

The present invention offers significant advantages over previously used methods for gene optimization. For example, DNA shuffling of codon-modified nucleic acids is not yet fully understood by the precise mechanism by which specific properties are mediated.
This may result in optimization of the desired properties. In addition, completely new properties can be obtained upon shuffling codon-modified DNA. That is, the shuffled DNA encodes a polypeptide or RNA that has the property of completely deleting the shuffled parental DNA. Thus, by altering the codon usage and / or the encoded amino acids of the related gene or other nucleic acid, molecular diversity is approached and the sequence gains the desired properties, including entirely novel properties. Can be shuffled.

In general, sequence recombination can be accomplished in many different formats and permutations of formats, as described in further detail below.

The targets for modification vary in various applications, as well as the properties sought to be obtained or improved. Examples of candidate targets for gaining or improving properties include genes encoding proteins having enzymatic or therapeutic or other commercially useful activities. A more extensive list is found above.
However, even this list is not intended to be limiting, as essentially any nucleic acid can be codon modified and shuffled using one or more of the processes herein. .

The shuffling method uses at least two mutant forms of the starting target (
A variant form can be, for example, a nucleic acid or a representation thereof, as a string in a computer program). The mutant form of the candidate codon-altered substrate is
Although they may exhibit substantial sequence or secondary structural similarity to each other, they should also differ in at least one and preferably at least two positions. Initial diversity between forms may be the result of natural variation. For example, different variant forms (homologs) can be obtained from different individuals or strains of an organism, or constitute related sequences (eg, allelic variants) from the same organism, or can be different organisms. Or an artificial homologue (eg, a codon-altered nucleic acid encoding the same or a similar protein). Any or all of these sequences may indicate or include a codon-altered nucleic acid.

[0051] Initial diversity may also be included. For example, the mutant form may be derived from the error-prone transcription of the first mutant form (eg, using error-prone PCR or a polymerase lacking proof-reading activity) (Liao (1990) Gen.
e 88: 107-111) or the first in the mutator strain.
(Mutator host cells are discussed in further detail below and are generally well-known). The initial diversity between the substrates is greatly increased in subsequent steps of the recombination for the generation of the library.

The mutator strain can contain any mutation in any organism that has impaired the function of mismatch repair. These include mutS, mutT, mutH, mutL,
Mutant gene products such as ovrD, dcm, vsr, umuC, umuD, sbcB, recJ and the like can be mentioned. Defects are achieved by genetic mutation, allelic replacement, selective inhibition by added reagents (eg, small compounds or expressed antisense RNA), or other techniques. The defect may be of the described gene or of a homologous gene in any organism. The properties or characteristics that can be obtained or improved can vary widely and, of course, depend on the choice of substrate.

At least two mutated forms of the nucleic acid (eg, they can confer the desired activity or they can be recombined to produce the desired activity) can be used to create a library of recombinant nucleic acids. Recombined to produce. The library is then screened to identify at least one recombinant nucleic acid that is optimized for the particular property or properties of interest.

Often, improvements are achieved after a single round of recombination and selection. However, recursive sequence recombination can be used to achieve still further improvements in the desired properties or to produce new (or "different") properties. Recursive sequence recombination requires successive cycles of recombination to generate molecular diversity. That is, one skilled in the art creates a family of nucleic acid molecules that exhibit some sequence identity to one another, but differ due to the presence of mutations. In any given cycle, recombination can occur in vivo or in vitro, intracellularly or extracellularly. In addition, the diversity arising from recombination may be determined by applying known mutagenesis methods (eg, error-prone PCR or cassette mutagenesis) to either the substrate or the product for recombination. Increased in cycles. However, in general, a single DNA shuffling cycle of a codon-modified nucleic acid provides surprisingly efficient nucleic acid production. Thus, while a recursive approach for shuffling may be used, a single recombination cycle is also preferred. Typically, two, three, four, five, or even ten or more recombination cycles are performed, each cycle optionally including one or more selection steps.

A recombination cycle usually involves at least one cycle of screening or selection for molecules having the desired properties or characteristics. If the recombination cycle is performed in vitro, the recombination product (ie, the recombination segment)
Often, they are introduced into cells prior to the screening step. The recombinant segment can also be ligated to a suitable vector or other regulatory sequence before screening. Alternatively, recombinant products produced in vitro are often packaged in a virus (eg, bacteriophage) prior to screening.
If the recombination is performed in vivo, the recombination product can often be screened in the cell where the recombination occurs. In other applications, the recombinant segments are extracted from the cells and optionally packaged as viruses prior to screening.

The nature of the screening or selection depends on which property or feature is obtained,
Or that the improvement will vary depending on the property or characteristic that is considered, and many examples are discussed below. It is not necessary to understand the molecular criteria by which a particular product of recombination (recombinant segment) acquires new or improved properties or characteristics compared to the starting substrate. For example, a gene may have many component sequences,
Each has a different intended role (eg, coding sequence, regulatory sequence, targeting sequence, stabilizing sequence, subunit sequence, and sequence affecting aggregation). Each of these component sequences can be altered and recombined simultaneously. Screening / selection is then performed, for example, on recombinant segments with increased ability to confer activity on cells without having to contribute to such an improvement on any individual component sequence of the vector. Can be.

Depending on the particular screening protocol used for the desired properties, the first round or rounds of screening often involve bacterial cells due to high transfection efficiency and ease of culture. Can be done using However, bacterial expression is often infeasible or undesirable, and yeast, fungal, or other eukaryotic systems are also used for library expression and screening. Similarly, other types of screens, which are not sensitive to screening in bacterial or simple eukaryotic library cells, have been described for use in environments that are close to those of their intended use. Performed in selected cells. The last round of screening may be performed on the correct cell type for the intended use.

If further improvements in properties are desired, at least one and usually
The collection of recombinant segments surviving the first round of screening / selection is subjected to a further round of recombination. These recombinant segments can be recombined with each other or with an exogenous segment presenting the original substrate or a further variant thereof. Again, recombination can take place in vitro or in vivo. If the previous screening step identified the desired recombinant segment as a component of the cell, the component can be subjected to further recombination in vivo,
Alternatively, it may be subjected to further recombination in vivo or isolated before performing a round of recombination in vitro. Conversely, if the previous screening step identified the desired recombinant segments in a naked form or identified the desired recombinant components as components of the virus, these segments would have been used in rounds of recombination in vivo. Can be introduced into cells to perform The second recombination (independent of how it is performed) is the set resulting from the previous round (or from multiple previous rounds (eg, if the process is repeated multiple times)) An additional recombinant segment is generated that contains the additional diversity present in the recombinant segment.

The second round of recombination is followed by a further round of screening / selection according to the principles discussed above for the first round. The stringency of the screening / selection can be increased between rounds. Also, the nature of the screening and the properties being screened may be such that improvement in one or more properties is desired, or
If it is desired to acquire one or more new properties, it may change between each round. Further rounds of recombination and screening may then take place until the recombinant segment has evolved sufficiently to acquire the desired new or improved property or function.

The practice of the present invention involves the construction of recombinant nucleic acids and the expression of the gene in transfected host cells. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids, such as expression vectors, are well known to those of skill in the art.
General text describing molecular biology techniques useful herein include: Berger and Kimmel, Guide to
Molecular Cloning Technologies, Methods
in Enzymology, Volume 152, Academic Press,
Inc. , San Diego, CA (Berger); Sambrook et al.
Molecular Cloning-A Laboratory Man
ual (2nd edition), Volumes 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New Yo
rk, 1989 ("Sambrook"), and Current Prot
ocols in Molecular Biology, F.C. M. Ausub
el et al., Current Protocols, Greene Publis
hing Associates, Inc. And John Wiley & So
ns, Inc. , A joint venture (replenished in 1998) ("Aussub
el "). Methods for transducing cells, including plant and animal cells, using nucleic acids are generally available as methods for expressing proteins encoded by such nucleic acids. Berger, Ausubel, and Sambrook
In addition to k, general references useful for culturing animal cells include the following: Freshney (Culture of Animal Cell)
s, a Manual of Basic Technology, 3rd edition, Wi
ley-Liss, New York (1994)), and the references cited therein, Humason (Animal Tissue Techni).
ques, 4th edition, W.W. H. Freeman and Company (1979)
), And Ricciardelli et al., In Vitro Cell De.
v. Biol. 25: 1016-1024 (1989). References for cloning, culturing, and regeneration of plant cells include: Payn
e et al. (1992) Plant Cell and Tissue Culture.
e in Liquid Systems, John Wiley & Son
s, Inc. New York, NY (Payne); and Gamborg
And Phillips (eds.) (1995) Plant Cell, Tissue
e and Organ Culture; Fundamental Meth
ods, Springer Lab Manual, Springer-Ve
rlag (Berlin Heidelberg New York) (Gam
borg). Various cell culture media are available from Atlas and Parks (Ed.), Th.
e Handbook of Microbiological Media (
1993) CRC Press, Boca Raton, FL (Atlas). Further information about culturing plant cells can be found, for example, in available commercial literature such as: Life Science
ce Research Cell Culture Catalog (1
998), Sigma-Aldrich, Inc. (St Louis, MO
) (Sigma-LSRCCC), and also, for example, Sigma −
Aldrich, Inc (St Louis, MO) (Sigma-PCCS)
Plant Culture Catalog and Supplement (199)
7).

Those skilled in the art are familiar with in vitro amplification methods, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other RNA polymerase-mediated techniques (eg, NASBA). Examples of techniques that are sufficient for are found below: Berger, Sambrook, and Ausu
bel, ibid, and Mullis et al., (1987) U.S. Pat.
No. 202; PCR Protocols A Guide to Method
s and Applications (Edited by Innis et al.) Academic
Press Inc. San Diego, CA (1990) (Innis);
Arnheim and Levinson (October 1, 1990) C & EN 3
6-47; The Journal of NIH Research (199)
1) 3, 81-94; Kwoh et al. (1989) Proc. Natl. Acad.
Sci. USA 86, 1173; Guatelli et al. (1990) Proc.
Natl. Acad. Sci. USA 87, 1874; Lomell et al.
89) J. Amer. Clin. Chem 35, 1826; Landegren et al. (19
88) Science 241, 1077-1080; Van Brunt (1
990) Biotechnology 8, 291-294; Wu and Wal.
race, (1989) Gene 4,560; Barringer et al. (199
0) Gene 89, 117, and Sooknanan and Malek (
1995) Biotechnology 13: 563-564. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., US Pat. No. 5,426,039. An improved method of amplifying large nucleic acids by PCR is described in Cheng et al. (1994) Nature 369: 6.
84-685, and references therein. here,
PCR amplicons of up to 40 kb are generated. One skilled in the art will recognize that essentially any R
It is understood that NA can be converted to double stranded DNA suitable for restriction digestion, PCR extension, and sequencing using reverse transcriptase and polymerase. Aus
See bell, Sambrook, and Berger (all supra).

Oligonucleotides, for example, for use in in vitro amplification / gene reconstitution methods, for the use of gene probes, or as targets for shuffling (eg, synthetic genes or gene segments) are typically Are described, for example, in Beaucage and Caruthers (1981), Tetrahed.
ron Letts. , 22 (20): 1859-1862, for example, an automated synthesizer (eg, Needham-VanDeva).
(1984) Nucleic Acids Res. , 12: 615
9-6168 or as routinely practiced in the art) according to the solid phase phosphoramidite triester method. Oligonucleotides can also be made to order and ordered from a variety of commercial sources known to those of skill in the art. Purification of oligonucleotides to improve the quality of synthetic oligonucleotides (eg, using gel purification methods)
May be particularly desirable in the processes herein to improve the quality of the protocol for nucleic acid synthesis.

As described, essentially any nucleic acid can be obtained from The Midland Cer.
tied Reagent Company (mcrc@oligos.c)
om), The Great American Gene Company (
http: // www. genco. com), ExpressGen Inc
. (Www.expressgen.com), Operon Technology
offices Inc. (Alameda, CA), and can be custom ordered from any of a variety of commercially available sources, such as many other sources. Similarly, peptides and antibodies are available from PeptidoGenic (pkim@ccnet.com), H
TI Bio-products, inc. (Http: //www.htib
io. com), BMA Biomedicals Ltd (UK), Bi
oSynthesis, Inc. , And many other sources can be custom ordered from any of a variety of sources.

Libraries with Altered Codons and Amino Acids In the method of the present invention, a library of codon-altered nucleic acids can be created and recombined. Codon-altered nucleic acids can also include differences in the encoded amino acid sequences, which can be either conservative or non-conservative in nature. Codon-altered nucleic acids can be derived from a single parent amino acid sequence, or can be derived from a family of original sequences (eg, natural or synthetic homologous variants of a given sequence). The library can be present, for example, in a pool or aliquot of cells, a viral plaque, a pool or aliquot of enzymatically synthesized nucleic acid, or a pool of chemically synthesized nucleic acid. Methods for preparing nucleic acid libraries are described, for example, in Berger, Sambrook.
And Ausubel, supra, are available and taught. In one embodiment, the library, as used in the present invention, comprises a sequence of at least two nucleic acids. In a further embodiment, the library of the invention comprises at least 2, 5, 10, 100, 1000 or more nucleic acid sequences.

As applied to the present invention, libraries are typically constructed with a high percentage of altered codons compared to the original (eg, wild-type) nucleic acid. . The difference in codon usage for each of the codon-altered nucleic acids can be 50%, 75%, or even 90% or more compared to the original nucleic acid. This eliminates hybridization to the parent nucleic acid (and thereby inhibits recombination with the parent nucleic acid, a desired feature in certain embodiments discussed below).

In some embodiments of the present invention, codons are modified in members of a gene family to increase the degree of identity between members. In one such embodiment, the genes are homologous genes from different species. In such cases, the degree of nucleic acid identity may be lower than the degree of amino acid identity, at least in part due to differences in codon usage between species. In a further embodiment, a homologous gene refers to different members of a gene family in a single species. Such genes may encode functionally distinct members of the gene family. Nevertheless, it shares significant structural or functional similarity. In a preferred embodiment, homologous genes are reverse transcribed in nucleic acid sequences, and the nucleic acid sequences are modified to increase the level of identity between them. The nucleic acid having the altered sequence can then be synthesized in vitro. In certain preferred embodiments, the modified nucleic acid sequence is at least identical to each other as the original amino acid sequence.

Further sequence diversity is provided by generating nucleic acids with non-overlapping non-conservative substitutions in each of the codon-altered nucleic acids when compared to the first nucleic acid. This provides a reversion to the wild type upon recombination,
On the other hand, if necessary, it allows for the incorporation of non-conservative changes into the sequence in the event that the non-conservative changes result in a detectable improvement during screening.

A codon modification of one or more codon-altered nucleic acids that provides one or more different hydrophobic core residues for the encoded polypeptide, as compared to the original polypeptide, is also Provided. This core amino acid modification provides minor differences in the encoded protein, while altering the mutation spectrum of the resulting nucleic acid, thereby increasing sequence diversity.

In addition, due to the limitations of the translation machinery for a given cell, codon usage can result in the expressed sequence being shuffled between different organisms (eg, animal cells, plant cells, bacterial cells, etc.). May need to be changed for optimal expression.
This results in a nucleic acid that encodes the same protein, but that, after a typical form of point mutation, obtains a variant diversity that differs from the original form of the protein.

[0070] In one embodiment, a phage library is created and mut
S, mutT, mutH, mutL, ovrD, dcm, vsr, umuC, u
It is recombined in a mutator strain, such as a cell having a mutant or defective gene product, such as muD, sbcB, recJ. The defect is achieved by genetic mutation, allelic replacement, selective inhibition by added reagents (eg, small compounds or expressed antisense RNA), or other techniques. Libraries with a high multiplicity of infection (MOI) are used to infect cells, increasing the frequency of recombination. Additional strategies for making phage libraries and / or for recombination of DNA from donor and recipient cells are set forth in US Pat. No. 5,521,077. Further recombination strategies for recombination of plasmids in yeast are given in WO 97/07205.

The library created can be a set of molecules in vitro, or can be in a cell or phage. A hypothetical nucleic acid library generated in silico is also a feature of the invention (Serifonov and Stemme).
r, see also supra). Generally, the library is screened to identify at least one recombinant nucleic acid that exhibits different or improved activity as compared to the parent nucleic acid or the nucleic acid to be recombined. Further details on creating suitable libraries are found below, for example, in the section entitled "Formats for Sequence Recombination".

Codon Alterations and Targets for Shuffling Essentially any nucleic acid can be codon altered and shuffled. No attempt has been made herein to identify a number of known nucleic acids. General sequence preservation agencies for known proteins include GenBank, E.
MBL, DDBJ, and NCBI. Other repositories can be easily identified by searching the Internet.

One class of preferred targets for activation includes nucleic acids encoding therapeutic proteins such as: erythropoietin (EPO), insulin, peptide hormones (eg, human growth hormone) Growth factors and cytokines (eg, epithelial neutrophil activating peptide-78, GROα / MGSA, G;
ROβ, Groγ, MIP-1α, MIP-1β, MCP-1, epidermal growth factor,
Fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, interferon, interleukin, keratinocyte growth factor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF, pleiotropin, SCF, c-kit ligand, VEGEF, G-CSF, etc.). Many of these proteins are commercially available (see, for example, Sigma BioSciences 1997, catalogs and price lists), and the corresponding genes are well known.

Another class of preferred targets are activators of transcription and expression. Exemplary transcription and expression activators include genes and proteins that regulate cell growth, differentiation, regulation, and the like. Activators of expression and transcription are found in prokaryotes, viruses, and eukaryotes, including fungi, plants, and animals, including mammals, and provide a wide range of therapeutic targets. Activators of expression and transcription include, for example, binding to receptors, stimulating signaling cascades, regulating expression of transcription factors, binding to promoters and enhancers, binding to proteins that bind promoters and enhancers, unwinding DNA It is clear that transcription is regulated by a number of mechanisms, such as by pre-mRNA splicing, RNA polyadenylation, and RNA degradation. Expression activators include: cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products (eg, interleukins (eg, IL-1, IL-2, IL-8, etc.)), interferons , FG
F, IGF-I, OGF-II, FGF, PDGF, TNF, TGF-α, TG
F-β, EGF, KGF, SCF / c-Kit, CD40L / CD40, VLA
-4 / VCAM-1, ICAM-1 / LFA-1, and hyalulin (hyalin)
urin) / CD44; signaling molecules and the corresponding oncogene products (
For example, Mos, Ras, Raf, and Met); and transcriptional activators and suppressors (eg, p53, Tat, Fos, Myc, Jun, M.).
yb, Rel, and steroid hormone receptors (eg, estrogen,
Progesterone, testosterone, aldosterone, LDL receptor ligand, and corticosterone)).

Similarly, proteins for possible vaccine applications, derived from infectious organisms, are described in more detail below and include: infectious fungi (eg, Aspergillus, Candida species) Bacteria (especially E. coli, which serves as a model for pathogenic bacteria), as well as bacteria of medical interest (eg, S.
tapylococci (eg, aureus), Streptococci
i (for example, pneumoniae), Clostridia (for example, per
fringens), Neisseria (e.g., gonorrhoea),
Enterobacteriaceae (eg, coli), Helicob
actor (eg, pylori), Vibrio (eg, cholera)
e), Capylobacter (eg, jejuni), Pseudomo
nas (eg, aeruginosa), Haemophilus (eg,
influenzae), Bordetella (eg, pertussis)
), Mycoplasma (eg, pneumoniae), Ureapla
sma (eg, urealyticum), Legionella (eg,
pneumophila), Spirochetes (eg, Trepone)
ma, Leptospira, and Borrelia), Mycobacterte
ria (eg, tuberculosis, smegmatis), Acti
nomyces (eg, israelii), Nocardia (eg, a
steroides), Chlamydia (eg, trachomatis)
), Rickettsia, Coxiella, Ehrichichia, Roc
halimaea, Brucella, Yersinia, Fracisell
a, and Pasteurella; protozoa (eg, sporozoa (eg, Plasmodia), rhizopods (eg, Entamoeb)
a), and flagella (Trypanosoma, Leishmania, Tri)
chomonas, Giardia, etc.); viruses (eg, (+) RNA viruses (eg, poxviruses (eg, vaccinia), picornaviruses (eg, polio); togaviruses (eg, rubella);
Flaviviruses (eg, HCV); and coronaviruses), (
-) RNA viruses (eg, rhabdoviruses (eg, VSV); paramyxoviruses (eg, RSV); orthomyxoviruses (eg, influenza); bunyaviruses; and arenaviruses), dsDNA viruses (eg, Reoviruses), RNA to DNA viruses (ie, retroviruses (eg, HIV and HTLV, in particular)), and certain DNs
A to RNA virus (eg, hepatitis B virus).

Other proteins relevant to non-medical uses (eg, transcription inhibitors,
Or toxins of crop pests (eg, insects, fungi, tobacco plants, etc.)
It is a preferred target for shuffling. Industrially important enzymes such as monooxygenases, proteases, nucleases and lipases are also preferred targets. As an example, subtilisin can be evolved by shuffling the form of the codon-altered subtilisin gene (von der
Osten et al. Biotechnol. 28: 55-68 (1993)
Providing nucleic acids encoding subtilisins). Folding assisting proteins such as chaperones are also preferred.

Preferred known genes suitable for codon alterations and shuffling also include: α-1 antitrypsin, angiostatin, antihemolytic factors, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuresis Polypeptides, atrial peptides, CXC chemokines (e.g., T39765, N
AP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10,
GCP-2, NAP-4, SDF-1, PF4, MIG), calcitonin, CC
Chemokines (e.g., monocyte chemistry-induced protein-1, monocyte chemistry-induced protein-2, monocyte chemistry-induced protein-3, monocyte inflammatory protein-1α, monocyte inflammatory protein-1β, R
ANTES, I309, R83915, R91733, HCC1, T58847
, D31065, T64262), CD40 ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, I
Factor X, Factor VII, Factor VIII, Factor X, fibrinogen, fibronectin, glucocerebrosidase, gonadotropin, hedgehog proteins (eg, Sonic, Indian, Desert), hemoglobin (for blood substitutes, radioactive Hirudin, human serum albumin, lactoferrin, luciferase, neutrin, neutrophil inhibitor (NIF), bone morphogenetic protein, parathyroid hormone, protein A, protein G, relaxin, renin, salmon calcitonin , Salmon growth hormone, soluble complement receptor I, soluble I-CAM1, soluble interleukin receptor (I
L-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15)
, Soluble TNF receptor, somatomedin, somatostatin, somatotropin,
Streptokinase, superantigen (ie, Staphylococcal)
Enterotoxin (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE)
, Toxic shock syndrome toxin (TSST-1), desquamation (Exfoliati)
ng) Toxins A and B, pyrogenic exotoxins A, B, and C; art
ritises mitogen, superoxide dismutase, thymosin α1
, Tissue plasminogen activator, tumor necrosis factor β (TNFβ), tumor necrosis factor receptor (TNFR), tumor necrosis factor α (TNFα), and urokinase. Many other known encoding nucleic acids, such as those in Genebank ^™ , can be codon-altered and shuffled.

Genes with Redesigned and Chemically Synthesized Codon Usage as Starting Materials for Shuffling of Gene Families—Expanding the Diversity of DNA Shuffling The preferred genetic code among organisms is Homologous genes from different organisms can have significantly lower homology at the nucleic acid level than at the amino acid level, as there are from similar to very different. For example, genetic information for some bacterial species is high in GC content (up to 70%), while others have AT-rich (> 60%) codon usage. Thus, genes from different organisms can have, for example, 40-60% amino acid identity, but have only 25-35% nucleic acid identity. It is often desirable to increase such levels of nucleic acid identity in order to increase the ability to recombine homologous sequences and thereby increase the efficiency of family shuffling using the methods of the present invention. In other aspects, for example, when using vectors where it is desired to reduce the rate of recombination between the vector and the host DNA, thereby increasing the safety of the vector, It is actually preferred to reduce the recombination rate in the system. The following examples describe certain issues with shuffling codon-altered nucleic acids.

Alteration of Codon Usage to Increase Homology In one aspect, the protein sequence of a member of a gene family can be, for example, one of the preferred codon usage charts in any conventional DNA manipulation program (eg, , Wisconsin Package (R), SeqW
eb, OMIGA, SeqApp, SeqPup, MacVector, DNA
(Strider, GeneWorks, etc.). The choice of codon usage is often determined by the host in which the gene is expressed. After maximizing the percentage of DNA sequence identity, the gene is
It is chemically synthesized using a high-throughput oligonucleotide synthesizer, for example, in a 96-well format, optionally in combination with polymerase and / or ligase gene synthesis methods.

In general, the similarity of DNA sequences after such treatment is at least as high as amino acid similarity, but based on the random frequency of sequence identity for any given codon. (Compared to the situation for a naturally occurring gene, which is usually less conserved than the encoded polypeptide), at least about 10% to 15% more than amino acid identity Can be. In most cases, the minimum requirement for amino acid identity can be as low as about 35%, yet retain sufficient nucleic acid homology for standard recombination methods (see The oligonucleotide-mediated recombination methods do not require a high level of similarity to achieve recombination, as discussed in supra.) However, in some cases (eg, where conserved regions form clusters within a gene), the minimum amino acid identity may still be lower.

Example: Shuffling of EPO with Modified Codons Protein erythropoietin α (also known as EPO), Epo
Gen and Procrit are hematopoietic hormones that provide various benefits to patients suffering from anemia (eg, a common symptom of AIDS). EPO is produced as a pharmaceutical with worldwide sales of approximately $ 1 billion. Thus, proteins with EPO-like activity (and preferably excellent activity) are of substantial commercial interest.

FIG. 1 shows the sequence of a portion of the monkey EPO gene. It is similar to the human EPO gene. FIG. 2 shows an example of a codon-modified EPO nucleic acid (or "wobble" EPO gene). Generally, transversion rather than transposition mutation is performed, where possible. The purpose of this strategy is to maximally disrupt hybridization of the resulting gene with naturally occurring EPO. FIG. 3 shows an alignment of naturally occurring EPO.

This strategy is better regulated by applying standard rules of base pairing (eg, elimination of GC pairing and GC stacking) to maximize sequence disruption. In addition, conservative or non-conservative amino acid modifications may also be made (in some cases where multiple codon-altered nucleic acids are shuffled, revert to wild-type amino acids during shuffling). Enable you to
It is desirable to generate codon-altered nucleic acids with non-overlapping non-conservative substitutions). The size of the sequence space for nucleic acids encoding EPO is large, about 2.8 × 10 ⁸⁸ different sequences (about 10 ⁸⁰ particles in the region; It is physically impossible to create all of the sequences with As shown schematically in FIG. 4, if one of skill in the art considers only the most different fluctuating genes (ones that use another type of codon for leucine, arginine, and serine), then the 10 There is still a sequence space of ³⁸ sequences. The overall strategy is to synthesize a library of wobble genes, screen for expression and activity if desired, and DNA shuffle the desired genes (eg, by an iterative process).

It is an object to further evolve codon-altered nucleic acids. A strategy for evolving any gene of interest by shuffling with other homologous genes from nature, the designed gene (which incorporates a library of designed sequence variations), and the gene containing the mutation of interest It is. However, codon-altered nucleic acids may not be easily shuffled using these genes due to sequence differences; or they may not be desirable for other reasons (eg, natural May be patented or may comprise elements of patented). These difficulties depend on the desired amino acid variation (
For example, it can be avoided by synthesizing a codon-altered homologous nucleic acid that encodes the one found in the homologous gene), which reduces the set of codons in close proximity to the nucleic acid (s) to be recombined. Have (by which, for example,
Allowing hybridization during recombination).

For example, after identifying a homologue of interest (eg, that shown in FIG. 3 for EPO), codon-altered nucleic acids encoding the same protein are synthesized using similar codon choices. Standard family shuffling is then performed using the codon-modified nucleic acid. This is shown schematically for EPO in FIG.

[0086] Fluctuation variants of EPO are screened for expression, and receptor binding assays are then performed in an ELISA format using the human EPOr-Fc fusion. After selection of binding mutants, activity is measured as thymidine incorporation in the UT7-EPO (human bone marrow cell line) cell proliferation assay. The cells are
Treated with various concentrations of EPO variants for 2-3 days, after which they are
Incubate for 4 hours in the presence of 3-H thymidine and measure thymidine incorporation. Erickson-miller et al. (1997) Blood.
90: 2421 (for receptor binding assays), and Wen et al.
4) J.I. Biol. Chem. 269: 22839-22846 (for assays for thymidine incorporation).

Assays for selecting EPO may also be based, for example, on the ability of the EPO protein to stimulate blood cell proliferation (eg, in vitro or in vivo).

Example: Codon Shuffling of G-CSF Family shuffling can be used to improve diversity from genes in the library being screened. In addition, heuristic approaches to design (eg,
The degeneracy between the primary and tertiary structures of the protein (ie, many different primary structures encode proteins with very similar three-dimensional structures) Can be used to take advantage.

A heuristic approach to design creates a sequence space of putative mutants that are highly biased (compared to random mutagenesis) to encode proteins that preserve the original activity. Use to Methods such as high-throughput (HTP) screening and phage panning are used to identify members of the designed library with the desired activity. DNA shuffling is used to improve this population of active clones so as to better regulate the mutant, so that one of skill in the art can compare the naturally occurring protein with an equivalent or better protein. Also makes it possible to evolve variants with superior functions.

FIGS. 6 and 7 show some mammalian homologs of G-CSF. FIG. 8 shows the hydrophobic core residues of human G-CSF (darkened). FIG. 9 illustrates a strategy for evolving human G-CSF variants that differ highly in sequence. First, these genes, including all of the diversity of mammalian homologs of G-CSF, are synthesized (genes 1, 2, and 3, FIG. 8). These genes are shuffled, phage panned against the G-CSF receptor, and HTP screened for biological function (receptor activity). Active clones are repeatedly shuffled and, if necessary, screened to generate evolved variants that are comparable (or greater) in activity (in human cells) to the human gene.

Next, those skilled in the art will evolve variants with highly mutated hydrophobic cores. This is schematically illustrated in FIG. 8, and a specific strategy for performing a biological screen is schematically illustrated in FIG. It is expected that the best variants obtained after screening a library in which the hydrophobic core has been randomized may be less active than wild-type human G-CSF. This is because it is difficult to optimize activity first in such a procedure. Family shuffling is used to obtain optimized variants. This means
By synthesizing genes containing mammalian homolog diversity at all but the location of the hydrophobic core; however, they are synthesized in the context of an evolved non-wild-type hydrophobic core. Family shuffling is used to optimize for the new hydrophobic core.

This strategy works because there is a functionally similar hydrophobic core for the wild-type protein, consisting of amino acids that differ significantly from the wild-type protein.
This understanding is supported by recent experiments on model systems. For example,
53% of the randomized sequences for three residues in the hydrophobic core of the λ repressor are folded and biologically active (Lim and Sauer (
1991). Mol. Biol. 219: 359-376). The design of proteins by patterning polar and non-polar amino acids (here, the 24 residues in the hydrophobic core of a 4-helix bundle protein) was performed by Kamtek
Randomized by ar et al. (1993) Science 262: 1321. The folded α-helix protein was recovered from approximately 1% of the clones. Desjarrais and Handel (1995) Current Op
inion in Biotechnology 6: 460-466 is R
A mutant of op (another 4-helix bundle protein) was shown. here,
Four hydrophobic core residues have been randomized and active variants have been obtained.
Axe et al. (1996) PNAS 95: 5590-5594 retain biological activity in a library by randomizing 13 hydrophobic core residues in the enzyme barnase. 23% clones. (1996) PNAS 93: 12155-1.
2158 describes a variant of T4 lysozyme in which 10 residues in the hydrophobic core have been replaced by Met. This reveals that the hydrophobic core of this protein is very tolerant of displacement. Taken together, this experimental evidence in a model system indicates that the hydrophobic core of many proteins can be replaced with other hydrophobic residues that pack in a similar fashion resulting in an active protein.
This degeneracy is used to evolve new forms of native and codon-altered genes.

A related approach is to search a protein database for proteins having similar activity to the protein desired to be evolved. (1996) J. Denesyuk et al. Theor. Biol shows the result of a search such as this for G-CSF. LIF is a protein that folds very similarly. One skilled in the art can use LIF as a "scaffold" on which the residues of G-CSF required for activity are located. G-CSF "wig (tou
In the case of a LIF with “pee” ”, one skilled in the art family shuffles the LIF scaffold such that the wig obtains a variant that is presented in a completely biologically active form.

Another approach is to use a computerized method to generate a family of putatively functional variants. Dahiyat and Mayo, Science, recently described computer methods used to design proteins. Proteins are mimicked on a computer, often with the aid of genetic algorithms, and the subsets that are considered “fit” are actually synthesized and “analyzed”. These computer methods are becoming increasingly powerful. These are useful, for example, for estimating families of mutations on the surface of proteins that do not disrupt function. DNA shuffling can be used to optimize active clones obtained by design. To give an example of G-CSF, one of skill in the art will be able to design all putative functional variant families to obtain all the structure-function data (eg, Rei
Combined with Dhaar-Olsen (Biochemistry) recently reported alanine scan data for G-CSF), a computerized method can be used. One skilled in the art can, for example, design a family to have minimal DNA identity to a wild-type gene, taking into account design constraints.
This library is synthesized, passed through biological screening and / or selection (ie, panning for the G-CSF receptor), and active variants are obtained. DNA shuffling is then used to evolve these active variants with the desired level of function.

The G-CSF protein is displayed on phage and screened for binding to the human G-CSF receptor in an ELISA format. Mutants that bind to the receptor are selected in a high-throughput screen for receptor activation. This cell-based assay measures receptor activation via a reporter gene (eg, luciferase) that is activated by a G-CSF responsive construct containing a STAT binding element.
1. Transform cells (e.g., HepG2) with a G-CSF responsive reporter plasmid and use the G-CSF mutant with the codon shuffled.
Treat for 5 hours. The cells are then lysed and luciferase activity is measured. See also Tian et al., (1998) Science 281: 257-259.

Examples: Codon Shuffling of Alkaline Phosphatase Alkaline phosphatase is a reporter enzyme widely used for ELISA assays, protein fusion assays, and secreted forms as reporter genes for mammalian cells It is. A more active form of this enzyme is desired.

A codon-altered form of alkaline phosphatase was generated by PCR assembly using the oligos shown in FIG. The oligo map is shown in FIG. The procedure used was essentially that of Stemmer et al. (1994) Gene 1
64: 49-57. Briefly, the oligos are mixed 1: 1 at various dilutions and, for example, 94 ° C. (60 seconds), 94 ° C. (
30 sec), 50 ° C. (30 sec), PCR at 72 ° C. (30 sec), for example, 25-6
Zero cycles were performed to perform PCR assembly. Assembling the BIAP gene
Performed in a circular format and the gene fragment was purified. About 100,000 colonies were screened on LB / am plates (about 1/10 is wt plasmid). Approximately 1/10 showed a blue color above background. Plasmid DNA showed the correct insertion.

In general, screening petri dishes using a representative colorimetric assay for phosphatase activity can be used for screening. This has advantages: it is simple, high-throughput, and semi-quantitative. Screening of microtiter plates is also preferred. this is,
Colorimetric and quantitative, but additional equipment may be required for implementation.

Examples: Codon shuffling to reduce production of competent virus from vectors and to produce attenuated virus as immunogenic compositions and vaccines. , Poxviruses, adenoviruses (Ads), herpes viruses, and parvoviruses. Common viral vectors include mouse leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), human immunodeficiency virus (HIV), adenovirus, adeno-associated virus (AAV)
, Epstein-Barr virus, canarypox virus, bovine pox virus, and vaccinia virus. Retrovirus,
Adeno-associated virus, herpes virus, and adenovirus-based viral vectors are all used as gene therapy vectors for the introduction of therapeutic nucleic acids into cells of an organism by ex vivo and in vivo methods.

If a viral vector is used, packaging cells are generally used to prepare virions used to transduce target cells. In these vectors, the transactive gene is inactivated and "rescued" by the transcomplement to provide a packaged vector. This form of the trans-complement may be transferred by co-infection of the packaging cell with a virus or vector that supplies a function missing from a particular gene therapy vector in trans, or a virus integrated into the genome of the packaging cell Provided by using a cell line having the components (eg, 293 cells). For example, cells transduced with an HIV or mouse retroviral proviral sequence, which lacks a nucleic acid packaging site, produce the transactive component of the retrovirus, but the capsid produced It does not specifically incorporate retroviral nucleic acids therein, and thus produces little or no surviving virus.

If these transduced “packaging” cells are subsequently transduced with a vector nucleic acid lacking the coding region for retroviral transactivation function but containing a packaging signal, Vector nucleic acids are packaged in infectious virions. Numerous packaging cell lines that are useful for MoMLV-based vectors, such as PA317 (ATCC CRL 90
78), which expresses MoMLV core and envelope proteins (M
Iller et al. Virol. 65: 2220-2224 (1991))) are known in the art. Carroll et al. (1994) Journa.
l of Virology 68 (9): 6047-6051 describes the construction of a packaging cell line for the HIV virus. Mutual complements of incomplete HIV molecular clones are described, for example, in Lori et al., (1992) Journal.
f Virology 66 (9) 5553-5560.

There is a viral replication function present in the vector that is not provided by the trans-complement required for the replication of the vector. In HIV, this typically includes: TAR sequences (sequences required for HIV packaging),
RRE sequence (if the instability element of gag p17 gene is included), and sequence encoding polyprint lacto. HIV sequences containing these functions are:
5 'long terminal repeat (LTR) and 5' LT responsible for efficient packaging
A portion of the sequence downstream of R (ie, the major splicing donor site (“MSD
))) And from the 3 ′ LTR upstream and from the polyprint lacto 3 ′ L
Includes up to U3R section of TR. The packaging site (psi or Ψ site) is mainly located between the MSD site and the gag start codon (AUG) in the leader sequence.
Partially located adjacent to the 5 'LTR. Garzino-Demo et al. (19
95) Hum. Gene Ther. 6 (2): 177-184.
For a general description of the structural elements of the HIV genome, see Holmes et al.,
See PCT / EP92 / 02787.

[0103] Another common vector is based on adenovirus. Typically, a vector containing the adenovirus ITR (Gingeras et al. (1982) J. Biol.
Chem. 257: 13475-13491) are packaged, for example, in 293 cells, which provide many of the components needed for vector packaging.

Adeno-associated virus (AAV) utilizes a helper virus, such as an adenovirus or a herpes virus, to achieve a productive infection. In the absence of helper virus function, AAV integrates (site-specifically) into the genome of the host cell, but the integrated AAV genome has no pathogenic effect. The integration step is such that the AAV genome remains genetically intact until the host is exposed to appropriate environmental conditions (eg, a lytic helper virus), where the host reenters the lytic life cycle. Make it possible. Samulski (1993) Curre
nt Opinion in Genetic and Developmentmen
t3: 74-80 and references cited therein provide an overview of the life cycle of AAV. For a general overview of AAV and adenovirus, or herpes helper functions, see Berns and Bohensky (1
987) Advanced in Virus Research, Acade
mic Press. , 32: 243-306. The genome of AAV is described in Laughlin et al. (1983) Gene, 23: 65-73. AAV expression is described in Beaton et al. Virol. , 63: 4
450-4454. Generally, all parvoviruses (B1
9 and AAV), including the ITRs of the virus.
Located inside. Recombinant AAV vectors (rAAV vectors) deliver foreign nucleic acids to a wide range of mammalian cells (Hermonat and Muzykka (1984)).
Proc. Natl. Acad. Sci. USA 81: 6466-6470;
Tratschin et al. (1985) Mol Cell Biol 5: 3251.
-3260), integrated into the chromosome of the host (McLaughlin et al. (1988)
J. Virol. 62: 1963-1973) and shows stable expression of the transgene in cell and animal models (Flotte et al. (1993) Proc.
Natl. Acad. Sci. ScL USA 90: 10613-10617). rA
AV vectors can infect non-dividing cells (Podsak)
(1994) J. Off. Virol. 68: 5656-66; Flotte et al. (1994) Am. J. Respir. Cell Mol. Biol. 11: 5
17-521). Additional advantages of rAAV vectors include the deletion of an endogenous strong promoter, which allows downstream cellular sequences, the naked icosahedral capsid structure of the vector (which stabilizes the vector, and Avoids possible activation (which facilitates enrichment by common laboratory techniques).

One problem with pre-existing vector packaging strategies is that the vector to be packaged can recombine in trans with a nucleic acid that provides the packaging function, thereby producing a replication competent virus. . this is,
It can be a problem both when the vector is produced for therapeutic applications (eg, gene therapy) and during the production of the encoded component in vitro. The present invention provides a method for reducing or eliminating recombination between a nucleic acid encoding a transactive component and a vector nucleic acid encoding a packaging site.

In particular, the nucleic acid subsequences of the vector, which flank the modified or deleted elements provided in trans, are designed to eliminate hybridization to wild-type sequences. Modify codons. Since these sequences do not hybridize, they cannot recombine with the nucleic acid producing the transactive component. One further advantage of this approach is that the vector also cannot recombine with the live virus (eg, in a human body infected with the virus that packages the vector elements). As mentioned above, 2
Two types of gene therapy vectors are retroviruses (these are, for example, HIV-1
And vectors based on adenoviruses, which can be packaged by adenoviruses.

Alternatively, the codons of the nucleic acids encoding the transactive components can be modified such that they do not hybridize to the wild-type sequence. This also prevents recombination with the vector having the wild-type sequence, thereby preventing the replication and formation of replication competent virus.

After modification of the codons, the vector or transactive nucleic acid can be shuffled as described above, and the nucleic acid can be packaged or screened, if appropriate, for its ability to be packaged. .

It is likewise understood that codon modifications of the viral sequence have additional uses. Codon changes in viral sequences can result in viral attenuation, for example, due to alterations in regulatory sequences, changes in mRNA secondary structure, inadequate translation due to rare codon usage, and the like. Such "codon attenuated" viruses are pre-existing attenuated viruses, which are typically produced by successive passages in cells other than the normal host type for the virus. Have significant advantages over In particular, codon-attenuated viruses can encode a wild-type set of proteins, making them ideal as immunogenic compositions for producing antibodies or for use as vaccines It is assumed that Viral proteins can also be used in various diagnostic assays. For example, current standard diagnostic tests for HIV infection use tests for the presence of anti-HIV antibodies in the blood by probing with viral proteins.

Examples: Codon usage libraries for evolving functional variants using reduced recombination with native gene sequences--adenoviruses Adenoviruses are used, for example, in gene therapy. This is a common vector. The virus is typically modified to render it replication deficient. This can be achieved, for example, by deleting the E1 and E4 genes. E
The function of E1 and E4 is that the vector deleted for E1 and E4 is a ubiquitous human embryonic kidney cell line 293 (which has been uncharacterized incorporated into their genome, (With the adenovirus fragment providing the function missing in trans) can be supplied by the trans complement. Replication-deficient adenovirus vectors, although at a low, but clinically significant frequency, recombine, thereby resulting in replication competent adenovirus contamination of the vector preparation. This is a significant problem for the application of adenovirus-based gene therapy vectors because adenovirus has deleterious effects on health.

In the present invention, a codon usage library containing a sequence of several hundred bases to several kilobases adjacent to the adenovirus E1 and E4 genes is prepared. This library has a high degree of diversity from the native adenovirus consensus sequence,
At the same time, they are designed to incorporate a large degree of codon degeneracy, thereby allowing a large space of sequence diversity to be searched. The principle of the design is to obtain variants encoding the same or similar protein sequence, but many mismatches to the wild-type E1 and E4 sequences are found in the 293 genome. These mismatches strongly reduce the frequency of unwanted recombination with the transcomplementing gene. As a result, the engineered adenovirus vector or adenovirus helper vector, which packages the adenovirus sequence in trans, including the packaging sequence (the adenovirus or adeno-associated virus ITR),
Has a reduced level of recombination. This provides a lower rate of production of competent adenovirus, thereby making culture and production of such vectors more secure.

(Evolutionally Impaired Viruses Created by Altering Large Codon Usage as a General Approach to Vaccines--HIV) HIV-1 and HIV2 are genetically related and antigenically cross-reactive. Yes,
They share a common cellular receptor (CD4). For an overview of HIV infection, see Rosenberg and Fauci (1993) Fundamenta.
l Immunology, 3rd edition, Paul (eds.) Raven Press,
Ltd. See, New York (Rosenburg and Fauci 1) and the references cited therein. HIV-1 infection is epidemic worldwide, thereby causing a variety of immune system deficiencies related to what is commonly referred to as acquired immunodeficiency syndrome (AIDS). HIV type 2 (HIV-2)
Has been isolated from both healthy individuals and patients with AIDS-like illness (Andreasson et al., (1993) Aids 7.989-93; Cla).
vel et al. (1986) Nature, 324, 691-695; Gao et al.
92) Nature 358, 495-9; Harrison et al. (1991) J.
own of Acquired Immunity Deficiency
Syndromes 4, 1155-60; Kanki et al. (1992) Ame.
rican Journal of Epidemiology 136,89
5-907; Kanki et al. (1991) Aids Clinical Revi.
ew 1991, 17-38; Romeieu et al. (1990) Journal o.
f Acquired Immunity Definition Syndrom
es 3,220-30; Naucler et al. (1993) International.
nal Journal of STD and Aids 4, 217-21
Naucler et al., (1991) Aids 5, 301-4). HIV-2
Although AIDS cases have been identified primarily from West Africa, sporadic HIV-2 related cases of AIDS have been identified in the United States (O'Brien et al. (1991) Aids 5).
85-8) and elsewhere. HIV-2 probably becomes endemic in other areas over time, following a pathway of transmission similar to HIV-1 (Harrison et al. (1991) Journal of Acq.
uired Immunity Syndromes 4,
1155-60; Kanki et al. (1992) American Journal.
of Epidemiology 136, 895-907; Romieu et al. (1990) Journal of Acquired Immun Def.
ciency syndromes 3,220-30). Epidemiological studies
HIV-2 causes human disease with lower penetrance than HIV-1, and
IV-1 suggests a significantly longer clinical incubation period (at least 25 years, and possibly longer) compared to less than 10 years; Kanki et al. (1991) Aids Clinical Review.
1991, 17-38; Romieu et al. (1990) Journal of
Acquired Immunity Syndromes
3,220-30, and Travers et al. (1995) Science 2
68: 1612-1615.

The ability of the HIV virus population to rapidly point mutate and evade the immune response poses a particular challenge for vaccine design. Although the immune system has responded to the virus in a step-wise and co-evolutionary manner, the present invention provides large, faster evolutions to produce new vaccines that stimulate more effective immune responses. Provide a general approach to offer.

For example, during the HIV infection incubation period (which lasts for several years), low titers of HIV may result from high HIV replication rates in combination with efficient virus clearance by the immune system. In response to these selective forces, viral mutations that reduce the recognition and neutralization of the immune system by B and T cell responses are selected. Lukashov et al. (1995) J. Am. Virol. 69
: 6911-6916. During prolonged incubation times, these mutations accumulate and ultimately overwhelm immune system control. Ho et al. (
1995) Nature.

Live attenuated vaccines (typically produced by prolonging the growth of human viruses in animal cells) are vaccines for several diseases, including mumps, rubella, and measles Has proven to be useful. Attenuation is
It involves the slow accumulation of many mutations throughout the genome of the virus during the course of adaptation to growth in animal cells. When used to vaccinate humans, the attenuated virus grows only weakly and elicits a complex immune response that the virus cannot avoid. Mutations in the attenuated virus can in principle revert in the same stepwise manner as it undergoes to grow in culture.

The risk of reversion is highest in viruses with a high mutation rate, such as HIV-1, which makes this strategy dangerous under current technology for vaccine development. However, the protective effect against HIV-1 was not
It is noteworthy that it is observed after infection with two viruses, which is much less pathogenic than HIV-1. Thus, a protective effect against HIV can be achieved with live vaccines.

[0117] To reduce the risk of reversion, a number of mutations need to be accumulated in the virus. However, if too many mutations are present, the immune system will in effect recognize an attenuated virus, rather than a virus with a protective effect against it.

As provided herein, immunogenic compositions (eg, vaccines) containing a number of silent substitutions are made. In contrast to existing attenuated vaccines, such viruses have a native protein sequence and elicit essentially the same immune response as the corresponding wild-type virus (typically Can also incorporate one or several additional impossible mutations). Codon change is 2
Have two effects, both of which increase the capacity of the vaccine.

First, as with standard attenuated viruses, the growth of codon-altered viruses is due to the effects of codon changes on translation, regulatory sequences, mRNA folding, packaging, etc. Be attenuated. For example, regulation of HIV-1 envelope expression has been observed as a result of codon usage. Haas et al. (
1989) Current Biology 6 (3): 315-324.

Second, codon changes create defects in the evolution of the virus. As discussed above, codon modifications alter the mutation avoidance spectrum of the virus, thereby disrupting the evolutionary choice for a particular codon.

[0121] Six codon amino acids are optimal targets for codon changes. Serine, arginine, and leucine each have four codons and one group of two codons in an unrelated group. See FIG. AGY to TCX
Switching to all serine codons and vice versa results in a protein with an unaltered amino acid sequence. See also FIG. However, these groups of codons differ significantly in the spectrum of amino acids that occur upon point mutation. 78% of all possible point mutations at one codon for serine (TCA) give rise to different amino acids as compared to the point mutation obtained for the AGT codon for serine. See FIG. Viruses with hundreds of codon changes can statistically access a totally different mutation spectrum (ie, "cloud") compared to the wild-type virus, and Space. The overall strategy for producing an evolutionarily defective virus is further illustrated in FIGS. 14, 15, and 17. FIG. 16, panels AC show ser,
The results of a single mutation at different codons for arg and leu are shown.

[0122] Point mutations are important for viruses such as HIV-1 to stay in favor of the host immune system. The amino acid mutations required for virus evasion are probably not random. Wild-type codon usage has evolved to allow for optimal immune system evasion. Wild-type codon usage is probably preferred for mutations that present changes that evade the host immune system without adversely affecting the encoded protein (s). Although complex, this natural pattern of amino acid sequence changes in the natural virus in response to the host system is not random and is less predictable. Seeiler-Moise
iwitsch et al. (1994) Annu. Rev .. Genet. 28: 559-
See also 596.

All codon alterations for ser, arg, and leu in the 875 aa envelope polyprotein of HIV-1 (eg, strain MN) are 18
Affects 7 codons (22%), resulting in 561 mutations. See, for example, FIG. 18, panels AD. If you change all HIV proteins,
The number of mutations is more than three times greater. Construction of such a codon-modified virus,
This is simplified by recent advances in the synthesis of long DNA sequences, which allow the assembly of average-sized plasmids from 40-mer oligos in one step, with approximately 75% efficiency. Stemmer (1994) Nature 370 389-391.
checking ... See also FIG. 19 for a list of oligos in one application for the synthesis of HIV Env. While the synthesis of the envelope gene is sufficient, the synthesis of the whole HIV genome from the oligo can be performed by this method.

In practice, a favorable equilibrium between attenuation and evolutionary deficiencies can be achieved, for example, by shuffling wild-type and codon-altered sequence DNA (eg, Stemmer et al. (1)
995) Gene 164: 49-53), followed by selection of the resulting library of viruses that maintain moderate growth despite many codon changes.

The attenuation that can be obtained by this approach may be sufficient to obtain vaccines for most viruses, but for HIV-1, evolutionary defects are more important due to the high mutation rate of the virus. is there. Live vaccines are used only if they elicit a complex and strong immune response sufficiently to prevent infection with the wild-type virus. Live virus vaccines are typically more protective than single protein vaccines because of the difficulty in out-mutating T and B cell responses to multiple epitopes. Weak growth of live virus vaccines results in larger antigenic doses, and point mutations
Increases the complexity of the immune response. To assess vaccine performance, vaccine performance was assessed in M. nemestrina or chimpanzees using A.
Evaluated using SIV variants known to produce IDS. An exemplary SIV sequence, SIVsmm, has Gene Bank accession number x143.
07. This virus is closely related to HIV-2. Hi
rsch (1989) Nature 339: 389-392. In general, the many complete sequences of HIV, SIV, and many other viruses are
It is found in well-known sequence repositories, including GenBank, EMBL, DDBJ, and NCBI. As a well characterized HIV clone, HI
V-1NL43, HIV-1SF2, HIV-1BRU, and HIV-1MN
Is mentioned. For an overview on the genetic diversity of HIV, see
er-Moiseiwitsch et al. (1994) Annu. Rev .. Genet
. 28: 559-96 and the references cited therein.

Several HIV-2 isolates (three molecular clones of HIV-2 (HIV-2 _ROD , HIV-2_SBL-ISYAnd HIV-2_UC1)), But also Makaki (
M. mulatta and M.M. nemstrina) or baboons
(Franchini et al. (1989) Proc. Natl.
Acad. Sci. U. S. A. 86, 2433-2437; Barnett et al.
(1993) Journal of Virology 67, 1006-14.
Boeri et al. (1992) Journal of Virology 66;
4546-50; Castro et al. (1991) Virology 184,21.
9-26; Francini et al. (1990) Journal of Viro.
logic 64, 4462-7; Putkonen et al. (1990) Aids 4.
Putkonen et al. (1991) Nature 352, 436.
-8). HIV-2 molecular claw is a human pathogen that can infect small primates.
Has been developed for AIDS pathogen studies and for HIV-1 and HIV-2.
Provides an attractive model for drug and vaccine development.

Recently, HIV-2 is a potential vaccine candidate against HIV-1, which is more toxic, due to its long asymptomatic latency and its ability to protect against infection by HIV-1. (Travers et al. (1995) Scie
268: 1621-2615, and Cohen et al. (1995) Sci.
ence 268: 1566). In a nine-year study by Travers et al. (Ibid.) Of HIV-2 infected prostitutes in West Africa, HIV-2 infection was 7% lower in HIV-1 infection.
It was determined to cause a 0% reduction. Thus, codon-modified HIV
-2 virus can also be used as a live vaccine against both HIV-2 and HIV-1. In addition, because HIV-2 is less pathogenic in nature than HIV-1, it is a preferred virus for modification in addition to HIV-1.

Formats for Sequence Recombination The methods of the present invention provide for performing recombination (“shuffling”) and for individual genes, whole plasmids, or viruses, multigene clusters, or even whole genomes. It entails performing screening or selection to "evolve" (Stemmer (1995) Bio / Technology 13:54).
9-553). Repeat cycles of recombination and screening / selection can be performed to further evolve the nucleic acid of interest. Such techniques do not require the extensive analysis and calculations required by conventional methods for manipulating polypeptides. Shuffling allows for the recombination of a large number of mutations in a minimal number of selection cycles, as opposed to the natural pair-wise recombination event, such as occurs during sexual replication. Becomes Accordingly, the sequence recombination techniques described herein offer particular advantages in that they provide recombination between mutations in any or all of these, thereby permitting various combinations of mutations. Provides a very fast way to search for modalities that can produce the desired result. However, in some instances, structural and / or functional information is available, which is not required for sequence recombination, but provides an opportunity for technology modification.

Illustrative formats and examples of sequence recombination (eg, referred to as “DNA shuffling,” “early forced evolution,” or “molecular breeding”) are described by the present inventors and coworkers. Are described in the following patents and patent applications: US Pat. No. 5,605,793; PCT Application W filed on Feb. 17, 1995.
O95 / 22625 (Application No. PCT / US95 / 02126); 199
U.S. Patent Application Serial No. 08 / 425,684, filed April 18, 5; 1
US patent application Ser. No. 08 / 621,430, filed Mar. 25, 996; PCT application WO 97/20078, filed Apr. 18, 1996 (application no. PCT / US96 / 05480). PCT Application WO 97/35966, filed March 20, 1997; U.S. Patent Application Serial No. 08 / 675,502, filed July 3, 1996; September 27, 1996. No. 08 / 721,824, filed on Sep. 26, 1997, PCT Application WO 98/13487; filed on Jul. 15, 1998 by del Cardayre et al. Evolution of Whol
e Cells and Organisms by Recursive S
equipment Recombination ”Attorney Reference Number 018097
020720US (PCT / US99 / 15972, filed July 15, 1999); Stemmer, Science 270: 1510 (1995).
); Stemmer et al., Gene 164: 49-53 (1995);
mer, Bio / Technology 13: 549-553 (1995);
Stemmer, Proc. Natl. Acad. Sci. U. S. A. 91:
10747-10751 (1994); Stemmer, Nature 370.
: 389-391 (1994); Crameri et al., Nature Medic.
ine 2 (1) 1-3 (1996); and Crameri et al., Natur.
e Biotechnology 14: 315-319 (1996). Each of these is incorporated by reference in its entirety for all purposes.

Recombination procedures generally exhibit substantial sequence identity to one another (eg, at least about 30%, 50%, 70%, 80%, or 90% or more sequence identity), Begin with at least two substrates that differ from each other at a particular location.
For example, at least one codon-altered nucleic acid is recombined with one or more additional nucleic acids herein, which may also be a codon-altered nucleic acid. The differences between the nucleic acids to be recombined can be any type of mutation, such as substitutions, insertions, and deletions. Often, the various segments differ from each other at about 5-20 positions. For recombination to generate increased diversity compared to the starting material, the starting materials must differ from each other by at least two nucleotide positions. That is, if only two substrates are present, they must be at least two different positions. If three substrates are present, for example, one substrate may be different from the second substrate at a single location, and the second substrate may be different from the third substrate at a different single location. Can be different. The starting DNA segments can be natural variants of each other (eg, allelic variants or species variants). More typically, these are codon-altered nucleic acids from one or more homologous nucleic acid sequences. Segments can also be derived from non-alleles that show some degree of structural and usually functional correlation (eg, codon-altered nucleic acids from different but homologous genes within the superfamily). The starting DNA segments can also be derived variants of each other. For example, one DNA segment can be produced by error-prone PCR replication of another DNA segment, or by replacement of a mutagenic cassette. Derived mutants can also be prepared by growing one (or both) of the segments in a mutagenic strain. In these situations, strictly speaking, the second DNA segment is not a single segment, but is a large family of related segments. The different segments forming the starting material are often the same length or substantially the same length. However, this is not necessary, for example, if one segment can be a subsequence of another segment. A segment can be present as part of a longer molecule (eg, a vector) or can be in an isolated form.

The starting DNA segments are recombined by any of the sequence recombination formats provided herein to create a diverse library of recombinant DNA segments. Such libraries, from those having fewer than 10 members, 10 ^5, 10 ^9, 10 ^12, 10 ^15, up to those having a member of 10 ²⁰ more than or greater than one, the size Can vary widely. In some embodiments, the generated starting segments and recombinant libraries are essentially full-length coding sequences and any necessary regulatory sequences required for expression (eg, promoter and polyadenylation sequences). )including. In another embodiment, the recombinant DNA segments in the library can be inserted into a general vector that provides the necessary sequences for expression before performing the screening / selection.

Use of Restriction Enzyme Sites to Recombine Mutants In some situations, the use of restriction enzyme sites in nucleic acids to direct the recombination of mutations in a nucleic acid sequence of interest. Is advantageous. These techniques are particularly preferred in the evolution of fragments that cannot be easily shuffled by other existing methods due to the presence of related DNA or other primary sequence motifs of interest. These situations also include recombination formats in which it is preferable to retain the particular sequence that is not mutated. The use of restriction sites is also important for large fragments (typically longer than 10 kb) (eg, clusters of genes (eg,
This is preferred for shuffling which cannot be easily shuffled and "PCR amplified" because of their size)). Fragments of up to 50 kb have been reported to be amplified by PCR (Barnes, P.
rc. Natl. Acad. Sci. U. S. A. 91: 2216-2220
(1994)), but this can be a problem for fragments larger than 10 kb, so alternative methods are preferred for shuffling in the range of 10-50 kb and beyond. Preferably, the restriction endonucleases used are of the class II type (Sambrook, Ausubel and Berger, supra) and preferably those that produce non-palindromic cohesive end overhangs, such as AlwnI, SfiI or BstX1. is there. These enzymes generate non-palindromic ends that allow for efficient ordered reassembly with DNA ligase. Typically, a restriction enzyme (ie, an endonuclease)
The site was prepared using conventional restriction enzyme mapping techniques (Sambrook, Ausubel,
And Berger, supra), by analyzing sequence information about the gene, or by introducing the desired restriction site into the nucleic acid sequence by synthesis (
Ie, by silent mutation incorporation). For example, one or more codon-altered nucleic acids may be placed at a restriction enzyme site, for example, one or more nucleic acids of interest (eg, including a gene or gene cluster modified by recombination with a codon-altered nucleic acid). And can be recombined.

The DNA substrate molecule to be digested can be derived from DNA that is replicated in vivo (eg, a plasmid preparation), or synthetic (ie, eg, containing a restriction enzyme recognition site of interest (preferably, the terminal (A fragment of a PCR-amplified nucleic acid carrying the DNA). Typically,
At least two variants of the gene of interest (each having one or more mutations) and at least one of the variants incorporating the codon modification have been determined to cleave in the nucleic acid sequence of interest. Digested with at least one restriction enzyme.
The restriction fragments are then ligated using DNA ligase to generate a full-length gene with the shuffled region. The number of regions shuffled depends on the number of cleavages in the nucleic acid sequence of interest. Shuffled molecules can be introduced into cells as described above, and screened or selected for desired properties as described herein. The nucleic acid can then be isolated from a pool (library) or clone having the desired properties,
It can then be subjected to the same procedure until the desired degree of improvement is obtained.

[0134] In some embodiments, at least one DNA substrate molecule or fragment thereof is isolated and subjected to mutagenesis. In some embodiments, the pool or library of religated restriction fragments is subjected to mutagenesis or further recombination protocols before the digestion-ligation process is repeated. “Mutage” as used herein refers to such techniques known in the art as PCR mutagenesis, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and the like. Includes repetitive sequence recombination by any of the techniques described herein.

Reassembly PCR A further technique for recombining mutations in nucleic acid sequences is “reassembly PCR”.
Use This method can be used to assemble multiple segments that have been separately evolved in a full-length nucleic acid template, such as a gene. This technology is
The pool of advantageous variants is known by previous work, or any mutagenesis technique known in the art (eg, PCR mutagenesis, cassette mutagenesis, doped oligo mutagenesis, chemical This is done if it has been identified by screening for mutants that can be created by mutagenesis, or by growing a DNA template in vivo in a mutator strain. The boundaries defining the segments of the nucleic acid sequence of interest are preferably located in intergenic regions, introns, or regions of the gene that probably do not have the mutation of interest. Preferably, oligonucleotide primers (oligos) are synthesized for PCR amplification of a segment of the nucleic acid sequence of interest. As a result, the sequence of the oligonucleotide overlaps the junction of the two segments. The overlap region is typically about 10 to 100
The length of the nucleotide.

[0136] Each of the segments is amplified using such a set of primers. The PCR product is then "reassembled" according to the assembly protocol as discussed herein to assemble the randomly fragmented gene. Briefly, in an assembly protocol, PCR products are first purified from primers, for example, by gel electrophoresis or size exclusion chromatography. The purified products are mixed with each other and denatured for about 1-10 cycles in the presence of polymerase and deoxynucleoside triphosphate (dNTP) and an appropriate buffer salt without the presence of additional primers ("self-priming"). Subject to reannealing and extension. Subsequent PCR using primers flanking the gene is used to amplify the yield of the fully reassembled and shuffled gene. In some embodiments, the resulting reassembled gene is subjected to mutagenesis before the process is repeated.

In the present invention, oligos such as PCR primers may contain codon modifications when compared to the starting sequence. In addition, oligonucleotides can form the basis of a PCR concatamerization reaction where overlapping hybridized oligonucleotides are extended in one or more PCR amplification cycles. In this embodiment, the template nucleic acid is not required (although the template or a fragment thereof may be added to the amplification mixture, but this may aid in the final reassembly of the full-length gene). Further details regarding oligonucleotide gene reassembly methods are described, for example, in US Patent No. 60 / 118,813, OLIGONUCLE, filed February 5, 1999, by Crameri et al.
OTIDE MEDIATED NUCLEIC ACID RECOMBIN
ATION ", and Crameri et al., Filed June 24, 1999,
USN60 / 141,049 "OLIGONUCLEOTIDE MED
Found in "IATED NUCLEIC ACID RECOMBINATION".

In a further embodiment, a P for amplification of a segment of the nucleic acid sequence of interest is used.
The CR primer is used to introduce a mutation into a target gene as described below. Mutations at sites of interest in a nucleic acid sequence are identified by screening or selection, by sequencing homologs of the nucleic acid sequence, and the like. Oligonucleotide PCR encoding wild-type and mutant information at the site of interest
Primers are synthesized. These primers are then used in PCR mutagenesis to create a library of full-length genes encoding permutation of wild-type and mutant information at the designed positions. This technique typically requires that the screening or selection process be expensive or cumbersome compared to the cost of sequencing the mutant gene of interest and synthesizing mutagenic oligonucleotides. It is advantageous if present or impractical.

Site-directed mutagenesis (SDM) with oligonucleotides encoding homologous variants before shuffling In some embodiments of the invention, sequence information from one or more substrate sequences is Of a given "parent" sequence, followed by recombination during rounds of screening or selection. Typically, this is accomplished using techniques well known in the art using oligonucleotides encoding one or more mutations from one substrate and another substrate sequence (eg, a homologous gene) as a template. (Eg, Berger, Au
subbel, and Sambrook, supra). After screening or selection for the improved phenotype of interest, the selected recombinant (s) can be further evolved using iterative techniques. After screening or selection, site-directed mutagenesis can be performed again with another collection of oligonucleotides encoding homologous variants, and the above process is repeated until the desired properties are obtained.

If the difference between the two homologs is one or more single point mutations in the codon,
Degenerate oligonucleotides encoding sequences in both homologs can be used.
One oligonucleotide may contain many such degenerate codons, and one of ordinary skill in the art will appreciate that all permutations across this block of sequence (permutaio)
n) can be searched exhaustively.

If the homologous sequence space is very large, it may be advantageous to limit the search for a particular variant. Thus, for example, computer modeling tools (Lat
hrop et al. (1996) J. Am. Mol. Biol. , 255: 641-665) can be used to model each homologous mutation on the target protein and discard any mutations that are expected to significantly disrupt structure and function. The operation of a genetic algorithm in silico to generate and predict mutation events is described in 199
USSN 60/118854, Serifono, filed February 5, 9
v and Stemmer "METHODS FOR MAKING CHARA
CTER STRINGS, POLYNUCLEOTIDES & POLYPEP
TIDES HAVING DESIRED CHARACTERISTICS "
Found in

Oligonucleotide and In Silico Shuffling Formats As noted above, at least two additional related formats are useful in the practice of the present invention. The first one is called shuffling in "silico"
Utilizes computer algorithms that perform "virtual" shuffling using genetic manipulation devices in the computer. As applied to the invention, a string of codon-altered gene sequences is recombined in a computer system and the desired product is created, for example, by reassembly PCR or ligation of synthetic oligonucleotides. . Shuffling in silico,
USSN 60/118854, Serif, filed February 5, 1999.
onov and Stemmer "METHODS FOR MAKING CH
ARACTER STRINGS, POLYNUCLEOTIDES & POLY
PEPTIDES HAVING DESIRED CHARACTER ST
RINGS, POLYNUCLEOTIDES & POLYPEPTIDES
HAVING DESIRED CHARACTERISTICS ". Briefly, genetic engineering devices (algorithms that indicate a given genetic event, such as a point mutation, recombination of two strands of homologous nucleic acid, etc.), for example, align strings of nucleic acid sequences (standard alignment software) Or by manual inspection and alignment) and predicting the consequences of recombination, to model recombination or mutation events that may occur in one or more nucleic acids. The expected result of the recombination is used to produce the corresponding molecule, for example, by oligonucleotide synthesis and reassembly PCR.

A second useful format is referred to as “oligonucleotide-mediated shuffling”, in which related homologous nucleic acids (eg, as applied to the invention, have been modified with codons) Oligonucleotides that correspond to a family of synthetic nucleic acid homologous variants), which are recombined to yield a selectable nucleic acid. This format is described in USSN 60/118, filed February 5, 1999.
813, Crameri et al., "OLIGONUCLEOTIDE MEDIA.
TED NUCLEIC ACID RECOMBINATION ", and 1
Cra of USSN 60 / 141,049, filed June 24, 999,
Meri et al., “OLIGONUCLEOTIDE MEDIATED NUCL
EIC ACID RECOMBINATION ". Briefly, selected oligonucleotides are synthesized, ligated, and extended, typically in an extension reaction mediated by either a polymerase or a ligase. This technique can be used to recombine a homologous nucleic acid sequence, or even a codon altered nucleic acid sequence that is not homologous.

One advantage of oligonucleotide-mediated recombination is the ability to recombine homologous nucleic acids with low sequence similarity, or even non-homologous nucleic acids. In these low homology oligonucleotide shuffling methods, a set of one or more fragmented nucleic acids (eg, truncated codon-modified oligonucleotides or synthesized codon-modified oligonucleotides) But,
For example, they are recombined using a set of cross-family diversity oligonucleotides. Each of these cross-over oligonucleotides has a plurality of sequence diversity domains corresponding to a plurality of sequence diversity domains from homologous or heterologous nucleic acids having low sequence similarity. Fragmented oligonucleotides, which are derived by comparison to one or more homologous or heterologous nucleic acids, can hybridize to one or more regions of the crossing oligo, thereby reducing recombination. make it easier.

[0145] When recombining homologous nucleic acids, a set of gene-shuffling oligonucleotides of overlapping families is used, which is a comparison of homologous nucleic acids containing nucleic acids with one or more codons modified, followed by the corresponding oligonucleotides. (Derived by nucleotide synthesis) are hybridized and extended (eg, by reassembly PCR or ligation). Thereby, a population of recombinant nucleic acids is provided, which can be selected for a desired trait or property. The set of overlapping family shuffling gene oligonucleotides comprises a plurality of oligonucleotide member types having consensus region subsequences derived from a plurality of homologous target nucleic acids.

Typically, as applied to the present invention, the gene shuffling oligonucleotides of the family, which comprise one or more codon-altered nucleic acid (s), have sequence identity. It is provided by aligning homologous nucleic acid sequences to select for conserved regions of sex and regions of sequence diversity.
Multiple families of gene shuffling oligonucleotides corresponding to at least one region of sequence diversity are synthesized (either sequentially or simultaneously).

A set of fragments, or a subset of fragments used in an oligonucleotide shuffling approach, may be obtained by cleaving one or more homologous nucleic acids (eg, using DNase), or more usually, at least one fragment. It can be provided by synthesizing a set of oligonucleotides corresponding to multiple regions of one nucleic acid (typically, oligonucleotides corresponding to a full-length nucleic acid are provided as members of a set of fragments of a nucleic acid). In the shuffling procedure herein, these truncated fragments are combined with a family of gene shuffling oligonucleotides, for example, to produce one or more of the recombinant codon-modified nucleic acid (s). It can be used in recombination reactions.

A further oligonucleotide shuffling format is described in Crameri et al., “O
LIGNUCLEOTIDE MEDIATED NUCLEC ACID
RECOMBINATION ", (agent registration number 02-296-2US)
And Welch et al., "USE OF CODON VARI."
ED OLIGONUCLEOTIDE SYNTHESIS FOR SYN
"THETIC SHUFFLING", (Attorney Registration No. 02-1007). In particular, these applications provide 3-nucleotide based synthesis of degenerate oligonucleotides, thereby providing codon substitution during shuffling of oligonucleotides. Briefly, this procedure uses 3-, rather than standard 1-nucleotide synthesis, to synthesize oligos.
Utilizes nucleotide phosphoramidite chemistry. Since the codons are changed as units, the synthesis scheme for degenerate oligonucleotides is simple.

Additional In Vitro DNA Shuffling Formats In one embodiment for shuffling DNA sequences in vitro, the first substrate for recombination is from a different individual, strain, or species of organism. Pools of related sequences (eg, different variant forms), such as homologs, or related sequences from the same organism, such as allelic variants. The sequence can be DNA or RNA, and can be of various lengths depending on the size of the gene or DNA fragment to be recombined or reassembled. Preferably, the sequence is from 50 base pairs (bp) to 50 kilobases (kb).

The pool of related substrates is converted, for example, into overlapping fragments of about 5 bp to 5 kb or more. Often, for example, the size of the fragment is from about 10 bp to 1000 bp, and often the size of the DNA fragment is from about 100 bp to 500 bp. The conversion can be mediated by a number of different methods (eg, DNase I or RNase digestion, random shear, or partial restriction enzyme digestion) or by shrambling methods mediated by the oligonucleotides of the family discussed in crameli et al. Can be achieved by the synthesis of oligonucleotides as in For a discussion of protocols for nucleic acid isolation, manipulation, enzymatic digestion, etc.
See ambrook et al. and Ausubel (both supra). The concentration of fragments of a nucleic acid of a particular length and sequence is often 0% of the total nucleic acid weight.
. Less than 1%, or less than 1%. The number of different specific nucleic acid fragments in a mixture will usually be at least about 2, 10, 100, 500, or 1,000.
0 or more.

The mixed population of nucleic acid fragments can be prepared by any of a variety of techniques, including heating,
This step is at least partially converted to single-stranded form using chemical denaturation, including the use of DNA binding proteins, etc. (in oligonucleotide-mediated methods, this step is omitted). ). Conversion to form a single-stranded nucleic acid fragment and then reannealing can be accomplished by heating from about 80 ° C to 100 ° C, more preferably from 90 ° C to 96 ° C.
Transformation can also be performed by single-stranded DNA binding proteins (Wold (1997) Annu.
Rev .. Biochem. 66: 61-92) or the recA protein (e.g., Kiianitsa (1997) Proc. Natl. Aca).
d. Sci. U. S. A. 94: 7837-7840). The single stranded nucleic acid fragment having a region of sequence identity with another single stranded nucleic acid fragment is then regenerated by cooling from 20 ° C to 75 ° C, and preferably from 40 ° C to 65 ° C. Can be annealed.
Regeneration can be accelerated by the addition of polyethylene glycol (PEG), other volume exclusion agents or salts. 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 can be KCl or NaCl. The concentration of PEG is preferably between 0% and 20%, more preferably between 5% and 10%. Reannealing fragments can be from different substrates. The annealed nucleic acid fragment is a nucleic acid polymerase (eg, Taq or Klenow) and dNTP (ie, dA
(TP, dCTP, dGTP, and dTTP). If the region of sequence identity is large, Taq polymerase can be used with a reannealing temperature between 45-65 ° C. If the area of identity is small, K
Lenow polymerase can be used with a reannealing temperature between 20-30 <0> C. The polymerase can be added to random nucleic acid fragments before annealing, simultaneously with annealing, or after annealing.

The process of denaturation, regeneration, and incubation of overlapping fragments in the presence of a polymerase or ligase to create a collection of polynucleotides containing different permutations of the fragments often involves in vitro nucleic acid in vitro. Called shuffling. This cycle is repeated as many times as desired. Preferably, the cycle is repeated from 2 to 100 times, more preferably, the sequence is repeated from 10 to 40 times. The resulting nucleic acid is about 50 bp
From about 100 kb, preferably from 500 bp to 50 kb. The population shows variants of the starting substrate which show substantial sequence identity thereto, but also differ at some positions. The population has more members than the starting substrate. The population of fragments obtained by shuffling is used to transform host cells, optionally after cloning into a vector.

In one embodiment utilizing in vitro shuffling, the subsequences of the recombination substrate have a substantial fraction (typically at least 20 percent or more incompletely extended amplification). Product) under the conditions that result in the amplification of the full-length sequence. Another embodiment uses random primers to prime the complete template DNA to generate shorter than full-length amplification products. Amplification products (including incompletely extended amplification products) are denatured and subjected to at least one additional cycle of reannealing and amplification. This variation, in which at least one cycle of reannealing and amplification provides a substantial fraction of incompletely extended product, is called "stuttering (s
tutting). In subsequent rounds of amplification, the partially extended (less than full length) product reanneals to the template species associated with a different sequence and primes the extension. In another embodiment, conversion of the substrate into fragments can be achieved by partial PCR amplification of the substrate.

[0154] In another embodiment, the mixture of fragments is spiked with one or more oligonucleotides. Oligonucleotides can be designed to include pre-characterized mutations (eg, codon modifications) of the wild-type sequence, or sites of natural mutation between solids or species. Oligonucleotides also typically include sufficient sequence or structural homology flanking such variants or variations to allow for annealing with the wild-type fragment. The annealing temperature can be adjusted depending on the length of homology.

In a further embodiment, recombination is effected by altering the temperature by altering the temperature, eg, when DNA fragments from one template prime on homologous positions of related but different templates. Occurs in one cycle. The change in temperature is indicated by recA (see Kiianitsa (1997), supra), ra
d51 (Namsaraev (1997) Mol. Cell. Biol. 17:
5359-5368), rad55 (Clever (1997) E
MBOJ. 16: 2535-2544), rad57 (Sung).
(1997) Genes Dev. 11: 1111-1121);
Alternatively, it can be induced by the addition of other polymerases (eg, viral polymerase, reverse transcriptase) to the amplification mixture. Altering the temperature can also be increased by increasing the concentration of the DNA template.

Another embodiment utilizes at least one cycle of amplification. This can be done using a collection of overlapping single-stranded DNA fragments of related sequences, and different lengths. The fragment may be a single-stranded DNA phage (eg, M13 (Wang (1997) Biochemistry 36: 948).
6-9492). Each fragment hybridizes to the polynucleotide strand of the second fragment from the collection and primes its extension, thus forming a polynucleotide whose sequence has been recombined. In a further variation, a ssDNA fragment of variable length relative to a first DNA template can be used to generate Pfu, T
aq, Vent, Deep Vent, U1Tma DNA polymerase, or other DNA polymerases (Cline (1996) Nu
cleic Acids Res. 24: 3546-3551).
The single-stranded DNA fragment is used as a primer for a second Kunkel-type template. It is formed from circular ssDNA containing uracil. This results in multiple substitutions of the first template for the second template. Levich
Kin (1995) Mol. Biology 29: 572-577; Jung
(1992) Gene 121: 17-24.

In some embodiments of the present invention, the shuffled nucleic acids obtained by using the iterative recombination methods of the present invention are introduced into cells and / or organisms for screening. Shuffled genes are used for initial screening, e.g., bacterial cells, yeast cells, fungal cells, vertebrate cells,
It can be introduced into an invertebrate cell or a plant cell. Bacillus species, such as B. subtilis and E. coli, have genes shuffled therein, which provide convenient shuttling against other cell types (between these bacterial and eukaryotic cells). A variety of vectors are available for shuttle material at the site; appropriate bacterial cells capable of inserting and expressing it can be inserted into Sambrook, Ausubel, and Berger (see all supra))). There are two examples: Shuffled genes can be introduced into bacterial, fungal, or yeast cells, either by integration into chromosomal DNA or as plasmids.

Bacterial, plant, animal, and yeast systems are preferred in the present invention. For example, in one embodiment, shuffled genes can be introduced into plant or animal cells for production purposes (transgenic plants being an increasingly important source of industrial enzymes). Is understood) or can be introduced into plant or animal cells for therapeutic purposes. Thus, the transgene of interest can be modified in vitro using the repetitive sequence recombination method of the present invention, and be novel or novel in bacterial, eukaryotic cells, or whole eukaryotic organisms. It can be reinserted into cells for in vitro / in situ selection for improved properties.

In Vivo DNA Shuffling Format In some embodiments of the present invention, a DNA substrate molecule (eg, containing a codon modification relative to a wild-type sequence) is introduced into a cell. Here, cellular mechanisms direct their recombination. For example, a library of variants is constructed and screened or selected for variants with an improved phenotype by any of the techniques described herein.

The DNA substrate molecule encoding the best candidate can be recovered by any of the techniques described herein, then fragmented and used to transfect a plant host and improved. Screened or selected for the function performed. If further improvement is desired, the DNA substrate molecule may be, for example, P
Recovered from the host cells by CR and the process is repeated until the desired level of improvement is obtained. In some embodiments, the fragment is denatured and re-annealed prior to transfection,
Recombination either coated with stimulated protein such as, or Noe in conjunction with a selectable marker such as ^R could be co-transfected, positive of cells receiving recombined versions of the gene of interest Enable selection. Methods for shuffling in vivo are described, for example, in PCT Application No. WO
98/13487, and WO 97/20078. The efficiency of shuffling in vivo can be enhanced by increasing the copy number of the gene of interest in the host cell.

Whole Genome Shuffling In one embodiment, the selection methods herein are utilized in a “whole genome shuffling” format. Extensive guidance on many forms of whole genome shuffling can be found in PCT / US99 / by del Cardayre et al.
15972, "Evolution of Whole Cells and
Organisms by Recursive Sequence Rec
Ombination "is found in the pioneering application of the present inventors and their co-workers. Any set of codon-altered nucleic acids can be used to transform cells, which can then be shuffled in a whole genome format.

In brief, shuffling of the whole genome does not make any assumptions about whether the nucleic acid can confer the desired properties. Instead, a complete genome (eg, derived from a library of genomes or isolated from an organism)
The cells are shuffled and a selection protocol is applied to the cells. These genomes can be spiked with any desired set of nucleic acids, including codon-modified nucleic acids.

Assays Relevant assays for the selection of the desired properties of the codon-modified nucleic acid will depend on the application. Many assays are known for detecting the activity of proteins, receptors, ligands, cells and the like. Formats include binding to immobilized components, cell or organism viability, production of a reporter composition, and the like.

In the high throughput assays of the present invention, it is possible to screen up to thousands of different shuffled variants per day. In particular, each well of the multititer plate can be used to perform a separate assay, or if the effect of concentration or incubation time is observed,
A single mutant can be tested every 10 wells. Thus, a single standard microtiter plate can assay about 100 (eg, 96) reactions. 1
If 536 well plates are used, a single plate can easily assay from about 100 to about 1500 different reactions. It is possible to assay several different plates in a single day; assay screening of about 6,000 to 20,000 different assays (ie including different nucleic acids, encoded proteins, concentrations, etc.) It is possible to use the centralized system of the present invention. More recently, microfluidics (microf) for the manipulation of reagents
luidic) approach is described in, for example, Caliper Technology
es (Mountain View, CA).

In one aspect, the members of the library (eg, cells, viral plaques, spores, etc.) are transformed into individual colonies (or plaques).
Separated on solid medium. Automated colony harvester (eg, Q-bo
t, Genetix, U.S.A. K. ), Colonies or plaques are identified and picked, and up to 10,000 different mutants are inoculated into a 96-well microtiter dish containing two 3 mm glass balls / well . Q-bot does not pick the entire colony, but rather inserts pins through the center of the colony and samples a small sample of cells (or mycelium) and spores (or virus in plaque applications). The amount of time the pins are in the colony, the number of dips inoculating the culture medium, and the time the pins are in the medium each affect the size of the inoculum, and each can be controlled and optimized. The uniform process of Q-bot reduces human manual errors and increases the rate at which cultures are established (approximately 10,000 /
4 hours). These cultures are then shaken in a constant temperature and humidity controlled incubator. The glass balls in the microtiter plate serve to promote the formation of a homogeneous aeration of the cells and to promote the dispersal of fragments of the mycelium resembling the fermentor splash. Clones from the culture of interest can be cloned by limiting dilution. As also described above, the plaques or cells that make up the library can also be directly used for protein production, either by detecting hybridization, protein activity, binding of the protein to the antibody, etc. Can be screened.

The ability to detect subtle increases in the capacity of shuffled library members over the parental strain depends on the sensitivity of the assay. The chance of finding an organism that has improvement is increased by the number of individual variants that can be screened by the assay. Preliminary screening can be used to increase the number of mutants treated (eg, up to 10-fold) to increase the chance of identifying a pool of sufficient size. The purpose of the initial screen was to quickly identify variants with a product titer equal to or better than the parent strain (s), and to identify liquid cells for subsequent analysis. The transfer of only these mutants to the culture.

[0167] Many well-known robotic systems have also been developed for solution phase chemistry that are useful in assay systems. These systems are based on Takeda
Chemical Industries, LTD. (Symate II, Zymark Corporation, Hopk)
inton, Mass. Orca, Hewlett-Packard, Pal;
o Alto, Calif. ) (Which mimics manual synthesis operations performed by scientists), including automated workstations. Any of the above devices are suitable for use with the invention (eg, high-throughput screening for molecules encoded by codon-modified nucleic acids). The nature and improvement (if any) of these modifications to these devices such that they can be manipulated as discussed herein with reference to the incorporated system,
It will be apparent to those skilled in the relevant arts.

High throughput screening systems are commercially available (eg, Zymark Corp., Hopkinton, Mass .; Air Tec).
hnical Industries, Mentor, OH; Beckman
Instruments, Inc. Fullerton, CA; Precisi
on Systems, Inc. , Natick, MA, etc.). These systems typically include pipetting of all samples and reagents,
Automate the entire procedure, including dispensing the liquid, timing the incubation, and final reading of the microplate in the detection device (s) appropriate for the assay. These configurable systems provide high throughput and quick start-up, as well as a high degree of flexibility and customization.

Manufacturers of such systems provide a variety of high-throughput, detailed protocols. Therefore, for example, Zymark Corp. Is the transcription of the gene,
Technical journals are provided that describe screening systems for detecting modulation such as ligand binding. Microfluidic approaches to the manipulation of reagents are also described, for example, in Caliper Technologies (Mountain View).
, CA).

The optical images visualized (and optionally recorded) by cameras or other reading devices (eg, photodiodes and data storage devices) are further described herein as appropriate. In any of the embodiments described herein, the image is processed, for example, by digitizing and / or storing the image, and analyzing the image on a computer. A variety of commercially available peripherals and software are available for digitizing, storing, and analyzing digitized video or digitized optical images (e.g., PC (Intel) x86 or pentium chip-compatible DOS (R), OS2 (R), WINDOWS (R), W
Devices based on INDOW NT® or WINDOWS 95®), MACINTOSH® or UNIX® (eg using a SUN® workstation) computer).

One conventional system, in typical use in the art, has light from an assay device to a cooled charge-coupled device (CCD) camera.
A CCD camera includes an array of photographic elements (pixels). The light from the specimen is CC
It is imaged on D. Particular pixels corresponding to regions of the analyte (eg, individual hybridization sites on a row of biological polymers) are sampled to obtain a light intensity reading for each location. Multiple pixels are processed in parallel to increase speed. The devices and methods of the present invention are readily used to visualize any sample, for example, by fluorescence or night vision microscopy techniques.

Software elements for manipulating strings of characters corresponding to codon-modified nucleic acids can be used to direct the synthesis of oligonucleotides involved in shuffling of codon-modified nucleic acids. Aggregated systems containing these and other useful features (eg, high-throughput liquid control software, image analysis software, data decoding software, solutions from a source to a destination operably linked to a digital computer) Robotic fluid control armature for transporting, labeled input device (eg, computer keyboard) for inputting data to a digital computer for controlling high-throughput fluid movement by the robotic fluid control armature Image scanners and the like for digitizing the labeled signal from the components of the assay are features of the present invention.

In one aspect, the present invention relates to a computer or computer readable medium comprising a database having a string of codon-altered nucleic acid sequences that is at least two artificial homologs, and wherein the user has at least An integrated system is provided that includes a user interface that allows selective viewing of a single array of strings. As discussed throughout, various sequence database programs exist for aligning and manipulating sequences. Additionally, standard textbook operating software (eg, language processing software (eg, Microsoft Word® or Corel Wordperfect®)) and database software (eg, Microsoft Excel®, Corel Quattro Pro) (A spreadsheet program software such as a registered trademark) or database software (for example, Microsoft)
ft Access (registered trademark) or Paradox (registered trademark) operates a user interface (for example, Wind) in order to operate a string of characters.
It can be used in combination with a GUI in a standard operating system, such as an ows, Macintosh, or LINUX system. Specialized alignment programs, such as BLAST, can also be incorporated in the system of the invention for alignment of codon-altered nucleic acids (or corresponding feature strands).

In addition to the integrated system described above, the integrated system can also include an automated oligonucleotide synthesizer operably linked to a computer or a computer-readable medium. Typically, the synthesizer is programmed to synthesize one or more oligonucleotides comprising one or more subsequences of at least two artificially homologous codon modified nucleic acids. You.

Modifications may be made to the methods and materials as described herein above without departing from the spirit or scope of the invention as claimed, and the invention comprises Can be utilized for a number of different applications, including:

[0176] Shuffled codons involved in an iterative process have been modified by DN
Use of the integrated system for testing ADNA.

Assays, kits, or systems that utilize the use of any one of the selection strategies, materials, components, methods, or substrates have been described herein above. The kit optionally further comprises components of an assay, device, or system, such as for performing a method or assay, packaging materials, and the like.
Includes one or more containers.

In a further aspect, the present invention provides kits using the methods and devices herein. The kits of the invention optionally include one or more of the following: (1) shuffled codon-modified components as described herein; (2) Instructions for performing the methods described herein and / or for operating the selection procedures described herein;
(3) one or more assay components; (4) containers for holding nucleic acids or enzymes, other nucleic acids, transgenic plants, animals, cells, etc., (5) packaging materials, and (6) one or more The software immobilized in a computer-readable medium comprising a sequence corresponding to a character string of a nucleic acid in which the codons have been modified.

In a further aspect, the invention relates to any device or kit for performing any of the methods or assays herein and / or for performing any of the assays or methods herein. Provided is the use of any of the components or kits herein for use.

Although the above invention has been described in some detail for purposes of clarity and understanding, various changes in form and detail may be made without departing from the true scope of the invention. Will be apparent to those of skill in the art in light of this disclosure. For example,
All the techniques and materials described above can be used in various combinations. All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication or patent document was individually listed. Have been.

[Brief description of the drawings]

FIG. 1 is the nucleic acid / amino acid sequence of a portion of the monkey EPO gene, similar to the human EPO gene.

FIG. 2 shows an example of a codon altered EPO nucleic acid sequence.

FIG. 3 shows an alignment of naturally occurring EPO.

FIG. 4 is a schematic diagram of a fluctuation sequence space of human EPO.

FIG. 5 is a schematic diagram of a fluctuation sequence space of a mammalian EPO family.

FIG. 6 is a sequence alignment of G-CSF homologs with species information.

FIG. 7 is a sequence alignment of the G-CSF homolog, and differences were extracted.

FIG. 8 is a sequence alignment showing the hydrophobic core residues of human G-CSF (darkened).

FIG. 9 is a schematic diagram showing a shuffling strategy of G-CSF.

FIG. 10 is a list of oligos used to generate codon-altered alkaline phosphatase.

FIG. 11 is a map of the oligos used to generate codon-altered alkaline phosphatase.

FIG. 12 is a schematic of vaccination with an evolutionarily defective virus.

FIG. 13 is a schematic representation of various mutations resulting from various codon types for ser, arg, and leu.

FIG. 14 is a schematic of vaccination with an evolutionarily defective virus.

FIG. 15 shows an uncomplexed “mutant cloud” versus a complex “mutant cloud”.
FIG. 1 is a schematic diagram of vaccination with an evolutionarily defective virus.

FIG. 16, Panels AC show the results of a single mutation of various codons for ser, arg, and leu.

FIG. 17 is a schematic representation of the evolution of a protein with an expanded mutation spectrum.

FIG. 18, Panel A shows a codon modified form of Env.

FIG. 18, Panel A shows a codon modified form of Env.

FIG. 18, Panel B shows a codon modified form of Env.

FIG. 18, panel C shows a codon modified form of Env.

FIG. 18, panel C shows a codon modified form of Env.

FIG. 18, Panel C shows a codon modified form of Env.

FIG. 18, Panel D shows a codon-modified form of Env.

FIG. 18, Panel D shows a codon modified form of Env.

FIG. 19A is a list of oligos in one application for the synthesis of HIV Env.

FIG. 19B is a listing of oligos in one application for the synthesis of HIV Env.

──────────────────────────────────────────────────続 き Continued on the front page (31) Priority claim number 60 / 118,813 (32) Priority date February 5, 1999 (1999.2.5) (33) Priority claim country United States (US) ( 31) Priority claim number 60 / 141,049 (32) Priority date June 24, 1999 (June 24, 1999) (33) Priority claim country United States (US) (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, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ, MD, RU , TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, 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, MD, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ , VN, YU, ZA, ZW (72) Inventor Stemmer, Willem P .; C. United States California 95030, Los Gatos, Cathy Court 108 F Term (Reference) 4B024 AA20 BA11 BA21 BA30 BA32 BA35 CA06 DA02 DA06 EA02 GA11 HA01 HA11 4B065 AA26X AA95X AA95Y AA96X AA96Y AB01 BA02 CA24 CA27 CA44 CA45 CA46

Claims (51)

[Claims] 1. A method for producing a codon-modified nucleic acid comprising the steps of: (i) providing a first nucleic acid sequence, wherein the nucleic acid sequence comprises a first polypeptide sequence; (Ii) providing a plurality of codon-altered nucleic acid sequences, each of which encodes the first polypeptide or an altered form thereof; and (iii) Recombining the plurality of codon-altered nucleic acid sequences to produce a target codon-altered nucleic acid, wherein the target codon-altered nucleic acid encodes a second protein. 2. The method of claim 2, wherein at least one of said plurality of codon-altered nucleic acid sequences comprises:
The method of claim 1, wherein the method does not hybridize to the first nucleic acid under stringent hybridization conditions. 3. The method of claim 1, wherein the first nucleic acid, or a nucleic acid comprising a subsequence comprising a substantially identical variant thereof, comprises one or more of the plurality of codon-modified nucleic acids or a target codon-modified nucleic acid. The method of claim 1, further comprising the step of shuffling with: 4. The method of claim 1, further comprising: (iv) screening said second protein for structural or functional properties. 5. The method of claim 1, further comprising: (iv) screening said second protein for structural or functional properties; and (v) said first protein. Comparing the structural or functional properties of the second protein to the structural or functional properties of the second protein. 6. The method of claim 1, wherein said second polypeptide has structural or functional properties that are equivalent to or better than said first polypeptide. 7. The method of claim 1, wherein said first and second polypeptides are homologous.
The method described in. 8. The method of claim 1, wherein said plurality of codon-altered nucleic acids comprises a library of codon-altered nucleic acids. 9. The method of claim 1, wherein said plurality of codon-altered nucleic acids comprises a library of codon-altered conservatively modified nucleic acids. 10. A library of codon-altered conservatively modified nucleic acids produced by the method of claim 9. 11. The method of claim 1, wherein the plurality of codon-altered nucleic acids comprises a library of codon-altered non-conservatively modified nucleic acids. 12. A library of codon-altered conservatively modified nucleic acids produced by the method of claim 11. 13. The method of claim 1, wherein said plurality of codon-altered nucleic acids is derived from a plurality of forms of a first nucleic acid. 14. The method of claim 1, wherein said plurality of codon-altered nucleic acid sequences comprises at least three codon-altered nucleic acids. 15. The method of claim 1, wherein the plurality of codon-altered nucleic acid sequences comprises one or more of the following structural features: (a) when compared to the first nucleic acid. 50% or more difference in codon usage for each of the codon-modified nucleic acids; (b) 75% or more difference in codon usage for each of the codon-modified nucleic acids when compared to the first nucleic acid. (C) a difference in codon usage of at least 90% for each of the codon-changed nucleic acids when compared to the first nucleic acid; (d) a codon change when compared to the first nucleic acid A difference in maximum codon usage for each of the modified nucleic acids; (e) non-overlapping non-conservative substitutions in each of the codon-modified nucleic acids when compared to the first nucleic acid; (f) one or more The codon-modified nucleic acid and A lack of high stringency hybridization with said first nucleic acid; and (g) one or more different hydrophobic cores for the encoded polypeptide when compared to said first polypeptide. Altering one or more codons of the codon altered nucleic acid to provide residues. 16. The method according to claim 1, wherein the percent identity between the second protein and the first protein is less than the percent identity between two of the plurality of codon-modified nucleic acids. The method described in. 17. The method of claim 1, wherein said first nucleic acid encodes a protein selected from: EPO, G-CSF, viral envelope protein, cytokine, and phosphatase. 18. The method according to claim 18, wherein the first nucleic acid sequence or the codon-modified nucleic acid sequence is
2. The method of claim 1, wherein the method is an isolated nucleic acid. 19. A target codon-altered nucleic acid produced by the method of claim 1. 20. The method of claim 1, wherein said first nucleic acid sequence or said codon altered nucleic acid sequence is a nucleic acid present in a cell. 21. A cell produced by the method of claim 20. 22. The method of claim 1, wherein each of said codon-altered nucleic acid sequences comprises a difference of at least two nucleotides when compared to said first nucleic acid. 23. The method of claim 1, further comprising introducing the target codon-altered nucleic acid into a cell or into a vector or virus. 24. A cell, vector, or virus produced by the method of claim 23. 25. The method of claim 1, wherein the target codon-altered nucleic acid is recombined with a portion of a viral genome to produce an attenuated virus. 26. An attenuated virus produced by the method of claim 25. 27. The method of claim 1, wherein said target codon-altered nucleic acid is recombined with a portion of a viral genome to produce an attenuated virus. The method wherein the attenuated virus produces an immune response upon infection by the virus in a mammal. 28. An attenuated virus produced by the method of claim 27. 29. The method of claim 1, wherein the target codon-altered nucleic acid is recombined with a portion of a retroviral genome to produce an attenuated retrovirus. The method wherein the attenuated retrovirus produces an immune response upon infection by the retrovirus in a mammal. 30. An attenuated retrovirus produced by the method of claim 29. 31. The method of claim 1, wherein the target codon-altered nucleic acid is recombined with a portion of a viral genome to produce a viral vector. 32. A viral vector produced by the method of claim 31. 33. The target codon-altered nucleic acid is recombined with a portion of a viral genome to produce a viral vector, wherein the vector requires transcomplementation for replication, and Wherein the vector has a reduced rate of reversion to its replicated form when compared to the corresponding viral vector lacking a subsequence corresponding to the target codon altered nucleic acid. The described method. 34. A viral vector produced by the method of claim 33. 35. The method of claim 33, wherein said vector comprises viral elements from one or more of the following: lentivirus, adenovirus, herpes virus, and adeno-associated virus. 36. A method for producing a library of codon-altered nucleic acids, comprising the steps of: (i) selecting a first nucleic acid sequence, wherein said nucleic acid sequence comprises a first nucleic acid sequence; And (ii) producing a plurality of codon-altered nucleic acid sequences, each of which encodes the first polypeptide or an altered form thereof. ,here,
The plurality of codon-altered nucleic acids comprises a library. 37. A codon-altered library produced by the method of claim 36. 38. The library of claim 37, wherein said library comprises at least two codon-altered nucleic acids. 39. The library of claim 37, wherein said library comprises at least 5 codon-altered nucleic acids. 40. The library of claim 37, wherein said library comprises at least 10 codon-altered nucleic acids. 41. The library of claim 37, wherein said library comprises at least 100 codon-modified nucleic acids. 42. The library of claim 25, comprising one or more of the containers, and explanatory material providing instructions for the method steps for recombination of two or more members of the library. kit. 43. The method of claim 41, further comprising recombining said plurality of codon-altered nucleic acids to produce a shuffled codon-altered library. 44. A codon-altered library produced by the method of claim 43. 45. The nucleic acid encodes a protein selected from EPO, cytokines, phosphatases, and viral envelope proteins.
37. The method of claim 36. 46. A composition comprising a plurality of codon-altered nucleic acids each encoding a first polypeptide or an altered form thereof. 47. A library of codon-altered nucleic acids comprising a plurality of codon-altered nucleic acids from a plurality of homologous nucleic acids. 48. The library of claim 47, wherein said plurality of codon-altered nucleic acids are recombined in vitro at an increased rate as compared to said plurality of homologous nucleic acids. 49. The level of identity in the plurality of codon-altered nucleic acids is at least as high as the level of identity in the plurality of polypeptides encoded by the plurality of homologous nucleic acids. A library according to claim 47. 50. Utilizing a computer, or a computer-readable medium, including a database having at least two artificially homologous codon-altered nucleic acid sequence strings, and utilizing one or more sequence strings in the database. An integrated system that includes a user interface that allows a person to selectively view. 51. The integrated system of claim 50, further comprising an automated oligonucleotide synthesizer operably linked to the computer or a computer-readable medium. , 1
A system programmed to synthesize one or more oligonucleotides, wherein the oligonucleotides comprise one or more subsequences of one or more of at least two artificially homologous codon-modified nucleic acids. .