U.S. patent application number 10/644410 was filed with the patent office on 2005-05-12 for saturation mutagenesis in directed evolution.
Invention is credited to Short, Jay M..
Application Number | 20050100985 10/644410 |
Document ID | / |
Family ID | 46281661 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100985 |
Kind Code |
A1 |
Short, Jay M. |
May 12, 2005 |
Saturation mutagenesis in directed evolution
Abstract
Disclosed is a rapid and facilitated method of producing from a
parentlal template polynucleotide, a set of mutagenized progeny
polynculeotides whereby at each original codon position there is
produced at least one substitute codon encoding each of the 20
naturally encoded amino acids. Accordingly, there is also provided
a method of producing from a parental template polypeptide, a set
of mutagenized progeny polypeptides wherein each of the 20
naturally encoded amino acids is represented at each original amino
acid position. The method provided is termed site-saturation
mutagenesis, or simply saturation mutagenesis, and can be used in
combination with other mutagenization processes, such as, for
example, a process wherein two or more related polynucleotides are
introduced into a suitable host cell such that a hybrid
polynucleotide is generated by recombination and reductive
reassortment. Also provided are vector and expression vehicles
incuding such polynucleotides, polypeptides expressed by the hybrid
polynucleotides and a method for screening for hybrid
polypeptides.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
DIVERSA CORPORATION
4955 DIRECTORS PLACE
SAN DIEGO
CA
92121
US
|
Family ID: |
46281661 |
Appl. No.: |
10/644410 |
Filed: |
August 20, 2003 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10644410 |
Aug 20, 2003 |
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10309587 |
Dec 4, 2002 |
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6764835 |
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10309587 |
Dec 4, 2002 |
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09756459 |
Jan 8, 2001 |
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6562594 |
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09756459 |
Jan 8, 2001 |
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09246178 |
Feb 4, 1999 |
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6171820 |
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09246178 |
Feb 4, 1999 |
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09185373 |
Nov 3, 1998 |
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6335179 |
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09185373 |
Nov 3, 1998 |
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08760489 |
Dec 5, 1996 |
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5830696 |
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09246178 |
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08962504 |
Oct 31, 1997 |
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6489145 |
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08962504 |
Oct 31, 1997 |
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08677112 |
Jul 9, 1996 |
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5965408 |
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08962504 |
Oct 31, 1997 |
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08651568 |
May 22, 1996 |
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5939250 |
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60008311 |
Dec 7, 1995 |
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60008316 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/91.2; 530/350 |
Current CPC
Class: |
C12N 9/00 20130101; C12N
15/1034 20130101; C07K 14/445 20130101; C12N 9/16 20130101; C12N
15/1027 20130101; C12N 15/1058 20130101; A61K 39/00 20130101; C12N
15/102 20130101; A61K 2039/53 20130101 |
Class at
Publication: |
435/069.1 ;
435/091.2; 435/320.1; 435/325; 530/350 |
International
Class: |
C12P 021/02; C12N
005/06; C12P 019/34; C07K 014/47 |
Claims
1. (canceled)
2. A method of providing a population of recombined nucleic acids,
the method comprising: (i) providing a population of
oligonucleotides comprising at least one set of codon-varied
oligonucleotides, wherein at least one member of the set of
codon-varied oligonucleotides is chemically synthesized using at
least trinucleotide sequences and wherein two or more members of
the population comprise overlapping oligonucleotides; (ii)
hybridizing at least two of the overlapping oligonucleotides to
each other to provide a population of hybridized overlapping
oligonucleotides, which population of hybridized overlapping
oligonucleotides comprises at least one codon-varied
oligonucleotide; and, (iii) elongating members of the population of
hybridized overlapping oligonucleotides, thereby providing the
population of recombined nucleic acids.
3. The method of claim 2, further comprising selecting at least
first and second nucleic acids to be recombined, wherein the set of
codon-varied oligonucleotides comprises a plurality of codon-varied
nucleic acids which correspond to the first and second nucleic
acids.
4. The method of claim 3, wherein the first and second nucleic
acids are homologous.
5. The method of claim 3, wherein the first and second nucleic
acids are non-homologous.
6. The method of claim 2, wherein the providing step comprises
trinucleotide synthesis comprising: (a) providing a substrate
sequence having a 5' terminus and at least one base, the 5'
terminus and at least one base having protecting groups thereon;
(b) removing the 5' protecting group of the substrate sequence to
provide a 5' deprotected substrate sequence; (c) coupling the 5'
deprotected substrate sequence with a selected trinucleotide
phosphoramidite sequence having a 3' terminus, a 5' terminus, and
three base groups, the 3' terminus, the 5' prime terminus, and the
three base groups having protecting groups thereon, thereby
yielding an extended oligonucleotide sequence; and, (d) repeating
steps (b) and (c), wherein the extended oligonucleotide sequence
yielded by each repeated step (c) becomes the substrate sequence of
the next repeated step (b) until a desired codon-varied
oligonucleotide is obtained.
7. The method of claim 2, wherein the providing step comprises
trinucleotide synthesis performed in an automated synthesizer which
automatically performs the steps of: (a) providing a substrate
sequence having a 5' terminus and at least one base, the 5'
terminus and at least one base having protecting groups thereon;
(b) removing the 5' protecting group of the substrate sequence to
provide a 5' deprotected substrate sequence; (c) coupling the 5'
deprotected substrate sequence with a selected trinucleotide
phosphoramidite sequence having a 3' terminus, a 5' terminus, and
three base groups, the 3' terminus, the 5' prime terminus, and the
three base groups having protecting groups thereon, thereby
yielding an extended oligonucleotide sequence; and, (d) repeating
steps (b) and (c), wherein the extended oligonucleotide sequence
yielded by each repeated step (c) becomes the substrate sequence of
the next step (b) until a desired codon-varied oligonucleotide is
obtained.
8. The method of claim 7, the method further comprising inputting
character string information into the automatic synthesizer
corresponding to the desired codon-varied oligonucleotides to be
obtained.
9. The method of claim 8, wherein the character string information
corresponds to two or more nucleic acids to be recombined.
10. The method of claim 2, wherein the providing step comprises
providing a substrate sequence having a 5' terminus and at least
one base, the 5' terminus and at least one base having protecting
groups thereon, the substrate sequence further comprising a 3' end
that is covalently attached to a solid support.
11. The method of claim 2, wherein the providing step comprises
coupling together one or more of: mononucleotides, trinucleotide
phosphoramidite sequences, and oligonucleotides.
12. The method of claim 2, wherein the providing step comprises
split-pool synthesis comprising: (a) providing substrate sequences,
each having a 5' terminus and at least one base, the 5' terminus
and at least one base having protecting groups thereon; (b)
removing the 5' protecting groups of the substrate sequences to
provide 5' deprotected substrate sequences; (c) coupling the 5'
deprotected substrate sequences with a population of a selected
trinucleotide phosphoramidite sequence, each having a 3' terminus,
a 5' terminus, and three base groups, the 3' terminus, the 5'
terminus, and the three base groups having protecting groups
thereon, thereby yielding extended oligonucleotide sequences; (d)
repeating steps (b) and (c), wherein the extended oligonucleotide
sequences yielded by each step (c) become the substrate sequences
of the next step (b), until extended intermediate oligonucleotide
sequences are produced; (e) splitting the extended intermediate
oligonucleotide sequences into two or more separate pools; (f)
removing the 5' protecting groups of the extended intermediate
oligonucleotide sequences to provide 5' deprotected extended
intermediate oligonucleotide sequences in the two or more separate
pools; (g) coupling the 5' deprotected extended intermediate
oligonucleotide sequences with one or more selected
mononucleotides, trinucleotide phosporamidite sequences, or
oligonucleotides in the two or more separate pools, thereby
yielding further extended intermediate oligonucleotide sequences;
(h) pooling the further extended intermediate oligonucleotide
sequences from the two or more separate pools into a single pool;
and, (i) repeating steps (b) through (h), wherein the further
extended intermediate oligonucleotide sequences in the single pool
of each step (h) become the substrate sequences of the next step
(b), until desired codon-varied oligonucleotides are obtained.
13. The method of claim 12, further comprising selecting at least
first and second nucleic acids to be recombined, wherein the set of
codon-varied oligonucleotides comprises a plurality of codon-varied
nucleic acids which correspond to the first and second nucleic
acids wherein the first and second nucleic acids are non-homologous
and are synthesized using the split-pool synthesis format.
14. The method of claim 13, wherein the non-homologous first and
second nucleic acids are less than 90 percent identical.
15. The method of claim 14, wherein the split-pool synthesis format
is module-based with a smallest module being a single trinucleotide
in length and a larger module being at least 15 nucleotides in
length.
16. The method of claim 2, wherein the providing step comprises
split-pool synthesis performed in an automated synthesizer which
automatically performs the steps of: (a) providing substrate
sequences having a 5' terminus and at least one base, the 5'
terminus and at least one base having protecting groups thereon;
(b) removing the 5' protecting groups of the substrate sequences to
provide 5' deprotected substrate sequences; (c) coupling the 5'
deprotected substrate sequences with a population of a selected
trinucleotide phosphoramidite sequence having a 3' terminus, a 5'
terminus, and three base groups, the 3' terminus, the 5' terminus,
and the three base groups having protecting groups thereon, thereby
yielding extended oligonucleotide sequences; (d) repeating steps
(b) and (c), wherein the extended oligonucleotide sequences yielded
by each step (c) become the substrate sequences of the next step
(b), until extended intermediate oligonucleotide sequences are
provided; (e) splitting the extended intermediate oligonucleotide
sequences into two or more separate pools; (f) removing the 5'
protecting groups of the extended intermediate oligonucleotide
sequences to provide 5' deprotected extended intermediate
oligonucleotide sequences in the two or more separate pools; (g)
coupling the 5' deprotected extended intermediate oligonucleotide
sequences with one or more selected mononucleotides, trinucleotide
phosporamidite sequences, or oligonucleotides in the two or more
separate pools, thereby yielding further extended intermediate
oligonucleotide sequences; (h) pooling the further extended
intermediate oligonucleotide sequences from the two or more
separate pools into a single pool; and, (i) repeating steps (b)
through (h), wherein the further extended intermediate
oligonucleotide sequences in the single pool of each step (h)
become the substrate sequences of the next step (b), until desired
codon-varied oligonucleotides are obtained.
17. The method of claim 16, further comprising selecting at least
first and second nucleic acids to be recombined, wherein the set of
codon-varied oligonucleotides comprises a plurality of codon-varied
nucleic acids which correspond to the first and second nucleic
acids wherein the first and second nucleic acids are non-homologous
and are synthesized using the split-pool synthesis format performed
in an automated synthesizer.
18. The method of claim 17, wherein the non-homologous first and
second nucleic acids are less than 90 percent identical.
19. The method of claim 18, wherein the split-pool synthesis format
performed in an automated synthesizer is module-based with a
smallest module being a single trinucleotide in length and a larger
module being at least 15 nucleotides in length.
20. The method of claim 16, the method further comprising inputting
character string information into the automatic synthesizer
corresponding to the desired codon-varied oligonucleotides to be
obtained.
21. The method of claim 20, wherein the character string
information corresponds to two or more nucleic acids to be
recombined.
22. The method of claim 16, wherein the providing step further
comprises providing a substrate sequence having a 3' terminus
covalently attached to a solid support.
23. The method of claim 2, wherein the hybridizing step occurs in
vitro.
24. The method of claim 2, wherein the hybridizing step occurs in
vivo.
25. The method of claim 2, wherein (iii) comprises elongating the
one or more members of the population of hybridized overlapping
codon-varied oligonucleotides with a polymerase.
26. The method of claim 25, wherein the polymerase is a
thermostable polymerase.
27. The method of claim 2, the method further comprising:
denaturing the population of recombined nucleic acids to provide a
set of denatured recombined nucleic acids; re-hybridizing at least
one member of the set of denatured recombined nucleic acids to at
least one other member of the set of denatured recombined nucleic
acids to provide a population of rehybridized recombined nucleic
acids; elongating one or more members of the population of
rehybridized recombined nucleic acids to provide a population of
further recombined nucleic acids; and, selecting at least one
member of the population of further recombined nucleic acids for at
least one desired trait or property.
28. The method of claim 2, the method further comprising the steps
of: denaturing the population of recombined nucleic acids to
provide a set of denatured recombined nucleic acids; re-hybridizing
at least one member of the set of denatured recombined nucleic
acids to at least one other member of the set of denatured
recombined nucleic acids to provide a population of rehybridized
recombined nucleic acids; elongating one or more members of the
population of re-hybridized recombined nucleic acids to provide a
population of further recombined nucleic acids; and, repeating the
denaturing, re-hybridizing and elongating steps at least once.
29. The method of claim 28, further comprising selecting at least
one member of the population of further recombined nucleic acids
for at least one desired trait or property.
30. The method of claim 28, wherein a plurality of members of the
population of recombined nucleic acids are selected for a desired
trait or property to provide first round selected nucleic acids,
the method further comprising: hybridizing at least one member of a
second set of overlapping codon-varied oligonucleotides to at least
one other member of the second set of overlapping codon-varied
oligonucleotides to provide a second population of hybridized
overlapping codon-varied oligonucleotides, which second set of
overlapping codon-varied oligonucleotides is derived from the first
round selected nucleic acids; and, elongating one or more members
of the second population of hybridized overlapping codon-varied
oligonucleotides to provide a second population of further
recombined nucleic acids.
31. The method of claim 30, further comprising sequencing the first
round selected nucleic acids, wherein the second set of overlapping
codon-varied oligonucleotides is derived from the first round
selected nucleic acids by aligning sequences of the first round
selected nucleic acids to one another to identify regions of
similarity and regions of diversity in the first round selected
nucleic acids, and synthesizing the second set of overlapping
codon-varied oligonucleotides to comprise a plurality of
oligonucleotides, each of which comprise subsequences corresponding
to at least one region of diversity.
32. The method of claim 30, wherein the first round selected
nucleic acids encode polypeptides of about 50 amino acids or
less.
33. The method of claim 30, wherein the second set of overlapping
codon-varied oligonucleotides comprises a plurality of
oligonucleotide member types which comprise consensus region
subsequences derived from a plurality of the first round selected
nucleic acids.
34. The method of claim 2, further comprising selecting at least
one member of the population of recombined nucleic acids for at
least one desired trait or property.
35. The method of claim 2, wherein the set of overlapping
codon-varied oligonucleotides comprises a plurality of
oligonucleotide member types which comprise consensus region
subsequences derived from a plurality of homologous target nucleic
acids.
36. The method of claim 2, wherein the set of overlapping
codon-varied oligonucleotides comprises at least 3 oligonucleotide
member types.
37. The method of claim 36, wherein the set of overlapping
codon-varied oligonucleotides comprises at least 5 oligonucleotide
member types.
38. The method of claim 37, wherein the set of overlapping
codon-varied oligonucleotides comprises at least 10 oligonucleotide
member types.
39. The method of claim 2, wherein the set of overlapping
codon-varied oligonucleotides comprises a plurality of homologous
oligonucleotide member types, wherein the homologous
oligonucleotide member types are present in equimolar amounts.
40. The method of claim 2, wherein the set of overlapping
codon-varied oligonucleotides comprises a plurality of homologous
oligonucleotide member types, wherein the homologous
oligonucleotide member types are present in non-equimolar amounts.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of protein engineering.
More specifically, this relates to a directed evolution method for
preparing a polynucleotides encoding polypeptide, which method
comprises the step of generating site-directed mutagenesis
optionally in combination with the step of polynucleotide
chimerization, the step of selecting for potentially desirable
progeny molecules (which may then be screened further), and the
step of screening the polynucleotides for the production of
polypeptide(s) having a useful property.
[0002] In a particular aspect, the present invention is relevant to
enzymes, particularly to thermostable enzymes, and to their
generation by directed evolution. More particularly, the present
invention relates to thermostable enzymes which are stable at high
temperature and which have improved activity at lower
temperatures.
BACKGROUND
[0003] Harvesting the full potential of nature's diversity can
include both the step of discovery and the step of optimizing what
is discovered. For example, the step of discovery allows one to
mine biological molecules that have industrial utility. However,
for certain industrial needs, it is advantageous to further modify
these enzymes experimentally to achieve properties beyond what
natural evolution has provided and is likely to provide in the near
future.
[0004] The process, termed directed evolution, of experimentally
modifying a biological molecule towards a desirable property, can
be achieved by mutagenizing one or more parental molecular
templates and idendifying any desirable molecules among the progeny
molecules. However, currently available technologies used in
directed evolution have several shortfalls. Among these shortfalls
are:
[0005] 1) Site-directed mutagenesis technologies, such as sloppy or
low-fidelity PCR, are ineffective for systematically achieving at
each position (site) along a polypeptide sequence the full
(saturated) range of possible mutations (i.e. all possible amino
acid substitutions).
[0006] 2) There is no relatively easy systematic means for rapidly
analyzing the large amount of information that can be contained in
a molecular sequence and in the potentially colossal number or
progeny molecules that could be conceivably obtained by the
directed evolution of one or more molecular templates.
[0007] 3) There is no relatively easy systematic means for
providing comprehensive empirical information relating structure to
function for molecular positions.
[0008] 4) There is no easy systematic means for incorporating
internal controls in certain mutagenesis (e.g. chimerization)
procedures.
[0009] 5) There is no easy systematic means to select for specific
progeny molecules, such as full-length chimeras, from among smaller
partial sequences.
[0010] Molecular mutagenesis occurs in nature and has resulted in
the generation of a wealth of biological compounds that have shown
utility in certain industrial applications. However, evolution in
nature often selects for molecular properties that are discordant
with many unmet industrial needs. Additionally, it is often the
case that when an industrially useful mutations would otherwise be
favored at the molecular level, natural evolution often overrides
the positive selection of such mutations when there is a concurrent
detriment to an organism as a whole (such as when a favorable
mutation is accompanied by a detrimental mutation). Additionally
still, natural evolution is slow, and places high emphasis on
fidelity in replication. Finally, natural evolution prefers a path
paved mainly by beneficial mutations while tending to avoid a
plurality of successive negative mutations, even though such
negative mutations may prove beneficial when combined, or may
lead--through a circuitous route--to final state that is
beneficial.
[0011] Directed evolution, on the other hand, can be performed much
more rapidly and aimed directly at evolving a molecular property
that is industrially desirable where nature does not provide
one.
[0012] An exceedingly large number of possibilities exist for
purposeful and random combinations of amino acids within a protein
to produce useful hybrid proteins and their corresponding
biological molecules encoding for these hybrid proteins, i.e., DNA,
RNA. Accordingly, there is a need to produce and screen a wide
variety of such hybrid proteins for a desirable utility,
particularly widely varying random proteins.
[0013] The complexity of an active sequence of a biological
macromolecule (e.g., polynucleotides, polypeptides, and molecules
that are comprised of both polynucleotide and polypeptide
sequences) has been called its information content ("IC"), which
has been defined as the resistance of the active protein to amino
acid sequence variation (calculated from the minimum number of
invariable amino acids (bits) required to describe a family of
related sequences with the same function). Proteins that are more
sensitive to random mutagenesis have a high information
content.
[0014] Molecular biology developments, such as molecular libraries,
have allowed the identification of quite a large number of variable
bases, and even provide ways to select functional sequences from
random libraries. In such libraries, most residues can be varied
(although typically not all at the same time) depending on
compensating changes in the context. Thus, while a 100 amino acid
protein can contain only 2,000 different mutations, 20.sup.100
sequence combinations are possible.
[0015] Information density is the IC per unit length of a sequence.
Active sites of enzymes tend to have a high information density. By
contrast, flexible linkers of information in enzymes have a low
information density.
[0016] Current methods in widespread use for creating alternative
proteins in a library format are error-prone polymerase chain
reactions and cassette mutagenesis, in which the specific region to
be optimized is replaced with a synthetically mutagenized
oligonucleotide. In both cases, a substantial number of mutant
sites are generated around certain sites in the original
sequence.
[0017] Error-prone PCR uses low-fidelity polymerization conditions
to introduce a low level of point mutations randomly over a long
sequence. In a mixture of fragments of unknown sequence,
error-prone PCR can be used to mutagenize the mixture. The
published error-prone PCR protocols suffer from a low processivity
of the polymerase. Therefore, the protocol is unable to result in
the random mutagenesis of an average-sized gene. This inability
limits the practical application of error-prone PCR. Some computer
simulations have suggested that point mutagenesis alone may often
be too gradual to allow the large-scale block changes that are
required for continued and dramatic sequence evolution. Further,
the published error-prone PCR protocols do not allow for
amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting
their practical application. In addition, repeated cycles of
error-prone PCR can lead to an accumulation of neutral mutations
with undesired results, such as affecting a protein's
immunogenicity but not its binding affinity.
[0018] In oligonucleotide-directed mutagenesis, a short sequence is
replaced with a synthetically mutagenized oligonucleotide. This
approach does not generate combinations of distant mutations and is
thus not combinatorial. The limited library size relative to the
vast sequence length means that many rounds of selection are
unavoidable for protein optimization. Mutagenesis with synthetic
oligonucleotides requires sequencing of individual clones after
each selection round followed by grouping them into families,
arbitrarily choosing a single family, and reducing it to a
consensus motif. Such motif is resynthesized and reinserted into a
single gene followed by additional selection. This step process
constitutes a statistical bottleneck, is labor intensive, and is
not practical for many rounds of mutagenesis.
[0019] Error-prone PCR and oligonucleotide-directed mutagenesis are
thus useful for single cycles of sequence fine tuning, but rapidly
become too limiting when they are applied for multiple cycles.
[0020] Another limitation of error-prone PCR is that the rate of
down-mutations grows with the information content of the sequence.
As the information content, library size, and mutagenesis rate
increase, the balance of down-mutations to up-mutations will
statistically prevent the selection of further improvements
(statistical ceiling).
[0021] In cassette mutagenesis, a sequence block of a single
template is typically replaced by a (partially) randomized
sequence. Therefore, the maximum information content that can be
obtained is statistically limited by the number of random sequences
(i.e., library size). This eliminates other sequence families which
are not currently best, but which may have greater long term
potential.
[0022] Also, mutagenesis with synthetic oligonucleotides requires
sequencing of individual clones after each selection round. Thus,
such an approach is tedious and impractical for many rounds of
mutagenesis.
[0023] Thus, error-prone PCR and cassette mutagenesis are best
suited, and have been widely used, for fine-tuning areas of
comparatively low information content. One apparent exception is
the selection of an RNA ligase ribozyme from a random library using
many rounds of amplification by error-prone PCR and selection.
[0024] In nature, the evolution of most organisms occurs by natural
selection and sexual reproduction. Sexual reproduction ensures
mixing and combining of the genes in the offspring of the selected
individuals. During meiosis, homologous chromosomes from the
parents line up with one another and cross-over part way along
their length, thus randomly swapping genetic material. Such
swapping or shuffling of the DNA allows organisms to evolve more
rapidly.
[0025] In recombination, because the inserted sequences were of
proven utility in a homologous environment, the inserted sequences
are likely to still have substantial information content once they
are inserted into the new sequence.
[0026] The term Applied Molecular Evolution ("AME") means the
application of an evolutionary design algorithm to a specific,
useful goal. While many different library formats for AME have been
reported for polynucleotides, peptides and proteins (phage, lad and
polysomes), none of these formats have provided for recombination
by random cross-overs to deliberately create a combinatorial
library.
[0027] Theoretically there are 2,000 different single mutants of a
100 amino acid protein. However, a protein of 100 amino acids has
20.sup.100 possible sequence combinations, a number which is too
large to exhaustively explore by conventional methods. It would be
advantageous to develop a system which would allow generation and
screening of all of these possible combination mutations.
[0028] Some workers in the art have utilized an in vivo site
specific recombination system to generate hybrids of combine light
chain antibody genes with heavy chain antibody genes for expression
in a phage system. However, their system relies on specific sites
of recombination and is limited accordingly. Simultaneous
mutagenesis of antibody CDR regions in single chain antibodies
(scFv) by overlapping extension and PCR have been reported.
[0029] Others have described a method for generating a large
population of multiple hybrids using random in vivo recombination.
This method requires the recombination of two different libraries
of plasmids, each library having a different selectable marker. The
method is limited to a finite number of recombinations equal to the
number of selectable markers existing, and produces a concomitant
linear increase in the number of marker genes linked to the
selected sequence(s).
[0030] In vivo recombination between two homologous, but truncated,
insect-toxin genes on a plasmid has been reported as a method of
producing a hybrid gene. The in vivo recombination of substantially
mismatched DNA sequences in a host cell having defective mismatch
repair enzymes, resulting in hybrid molecule formation has been
reported.
SUMMARY OF THE INVENTION
[0031] This invention relates generally to the field of nucleic
acid engineering and correspondingly encoded recombinant protein
engineering. More particularly, the invention relates to the
directed evolution of nucleic acids and screening of clones
containing the evolved nucleic acids for resultant activity(ies) of
interest, such nucleic acid activity(ies) &/or specified
protein, particularly enzyme, activity(ies) of interest.
[0032] This invention relates generally to a method of: 1)
preparing a progeny generation molecule (including a molecule that
is comprised of a polynucleotide sequence, a molecules that is
comprised of a polypeptide sequence, and a molecules that is
comprised in part of a polynucleotide sequence and in part of a
polypeptide sequence), that is mutagenized to achieve at least one
point mutation, addition, deletion, &/or chimerization, from
one or more ancestral or parental generation template(s); 2)
screening the progeny generation molecule--preferably using a high
throughput method--for at least one property of interest (such as
an improvement in an enzyme activity or an increase in stability or
a novel chemotherapeutic effect); 3) optionally obtaining &/or
cataloguing structural &/or and functional information
regarding the parental &/or progeny generation molecules; and
4) optionally repeating any of steps 1) to 3).
[0033] In a preferred embodiment, there is generated (e.g. from a
parent polynucleotide template)--in what is termed "codon
site-saturation mutagenesis"--a progeny generation of
polynucleotides, each having at least one set of up to three
contiguous point mutations (i.e. different bases comprising a new
codon), such that every codon (or every family of degenerate codons
encoding the same amino acid) is represented at each codon
position. Corresponding to--and encoded by--this progeny generation
of polynucleotides, there is also generated a set of progeny
polypeptides, each having at least one single amino acid point
mutation. In a preferred aspect, there is generated--in what is
termed "amino acid site-saturation mutagenesis"--one such mutant
polypeptide for each of the 19 naturally encoded
polypeptide-forming alpha-amino acid substitutions at each and
every amino acid position along the polypeptide. This yields--for
each and every amino acid position along the parental
polypeptide--a total of 20 distinct progeny polypeptides including
the original amino acid, or potentially more than 21 distinct
progeny polypeptides if additional amino acids are used either
instead of or in addition to the 20 naturally encoded amino
acids
[0034] Thus, in another aspect, this approach is also serviceable
for generating mutants containing--in addition to &/or in
combination with the 20 naturally encoded polypeptide-forming
alpha-amino acids--other rare &/or not naturally-encoded amino
acids and amino acid derivatives. In yet another aspect, this
approach is also serviceable for generating mutants by the use
of--in addition to &/or in combination with natural or
unaltered codon recognition systems of suitable hosts--altered,
mutagenized, &/or designer codon recognition systems (such as
in a host cell with one or more altered tRNA molecules).
[0035] In yet another aspect, this invention relates to
recombination and more specifically to a method for preparing
polynucleotides encoding a polypeptide by a method of in vivo
reassortment of polynucleotide sequences containing regions of
partial homology, assembling the polynucleotides to form at least
one polynucleotide and screening the polynucleotides for the
production of polypeptide(s) having a useful property.
[0036] In yet another preferred embodiment, this invention is
serviceable for analyzing and cataloguing--with respect to any
molecular property (e.g. an enzymatic activity) or combination of
properties allowed by current technology--the effects of any
mutational change achieved (including particularly saturation
mutagenesis). Thus, a comprehensive method is provided for
determining the effect of changing each amino acid in a parental
polypeptide into each of at least 19 possible substitutions. This
allows each amino acid in a parental polypeptide to be
characterized and catalogued according to its spectrum of potential
effects on a measurable property of the polypeptide.
[0037] In another aspect, the method of the present invention
utilizes the natural property of cells to recombine molecules
and/or to mediate reductive processes that reduce the complexity of
sequences and extent of repeated or consecutive sequences
possessing regions of homology.
[0038] It is an object of the present invention to provide a method
for generating hybrid polynucleotides encoding biologically active
hybrid polypeptides with enhanced activities. In accomplishing
these and other objects, there has been provided, in accordance
with one aspect of the invention, a method for introducing
polynucleotides into a suitable host cell and growing the host cell
under conditions that produce a hybrid polynucleotide.
[0039] In another aspect of the invention, the invention provides a
method for screening for biologically active hybrid polypeptides
encoded by hybrid polynucleotides. The present method allows for
the identification of biologically active hybrid polypeptides with
enhanced biological activities.
[0040] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a diagram illustrating the variety of likely
mutations in polypeptide that result from the introduction of
single point mutations in a polynucleotide encoding said
polypeptide by a method such as error-prone PCR. Because
replicative errors in a polynucleotide sequence, such as those
introduced using error-prone PCR are unlikely to generate two--much
less three--contiguous nucleotide changes, said methods are
unlikely to achieve more than 5-7 (on average) codon changes at
each codon position. Illustrated is the poor ability of this
approach for achieving all possible amino acid changes at each
amino acid site along the polypeptide. In contrast, the
site-saturation mutagenesis approach provided by the instant
invention does achieve a range of codon substitutions (preferably
comprising the 32 codons represented by the degenerate cassette
sequence N,N,G/T) so as to achieve all possible amino acid changes
at each amino acid site along a polypeptide.
[0042] FIG. 2 is a diagram illustrating the use of a
site-saturation mutagenesis approach for achieving all possible
amino acid changes at each amino acid site along the polypeptide.
The oligos used are comprised of a homologous sequence, a triplet
sequence composed of degenerate N,N, G/T, and another homologous
sequence. Thus, the degeneracy of each oligo is derived from the
degeneracy of the N,N, G/T cassette contained therein. The
resultant polymerization products from the use of such oligos
include all possible amino acid changes at each amino acid site
along the polypeptide, because the N,N, G/T sequence is able to
code for all 20 amino acids. As shown, a separate degenerate oligo
is used for mutagenizing each codon in a polynucleotide encoding a
polypeptide.
DEFINITIONS OF TERMS
[0043] In order to facilitate understanding of the examples
provided herein, certain frequently occurring methods and/or terms
will be described.
[0044] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds; an array of spatially
localized compounds (e.g., a VLSIPS peptide array, polynucleotide
array, and/or combinatorial small molecule array), biological
macromolecule, a bacteriophage peptide display library, a
bacteriophage antibody (e.g., scFv) display library, a polysome
peptide display library, or an extract made form biological
materials such as bacteria, plants, fungi, or animal (particular
mammalian) cells or tissues. Agents are evaluated for potential
activity as anti-neoplastics, anti-inflammatories or apoptosis
modulators by inclusion in screening assays described hereinbelow.
Agents are evaluated for potential activity as specific protein
interaction inhibitors (i.e., an agent which selectively inhibits a
binding interaction between two predetermined polypeptides but
which doe snot substantially interfere with cell viability) by
inclusion in screening assays described hereinbelow.
[0045] The term "amino acid" as used herein refers to any organic
compound that contains an amino group (--NH.sub.2) and a carboxyl
group (--COOH); preferably either as free groups or alternatively
after condensation as part of peptide bonds. The "twenty naturally
encoded polypeptide-forming alpha-amino acids" are understood in
the art and refer to: alanine (ala or A), arginine (arg or R),
asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or
C), gluatamic acid (glu or E), glutamine (gln or Q), glycine (gly
or G), histidine (his or H), isoleucine (ile or I), leucine (leu or
L), lysine (lys or K), methionine (met or M), phenylalanine (phe or
F), proline (pro or P), serine (ser or S), threonine (thr or T),
tryptophan (trp or W), tyrosine (tyr or Y), and valine (val or
V).
[0046] The term "amplification" means that the number of copies of
a polynucleotide is increased.
[0047] The term "antibody", as used herein, refers to intact
immunoglobulin molecules, as well as fragments of immunoglobulin
molecules, such as Fab, Fab', (Fab').sub.2, Fv, and SCA fragments,
that are capable of binding to an epitope of an antigen. These
antibody fragments, which retain some ability to selectively bind
to an antigen (e.g., a polypeptide antigen) of the antibody from
which they are derived, can be made using well known methods in the
art (see, e.g., Harlow and Lane, supra), and are described further,
as follows.
[0048] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0049] (2) An Fab' fragment of an antibody molecule can be obtained
by treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0050] (3) An (Fab').sub.2 fragment of an antibody can be obtained
by treating a whole antibody molecule with the enzyme pepsin,
without subsequent reduction. A (Fab').sub.2 fragment is a dimer of
two Fab' fragments, held together by two disulfide bonds.
[0051] (4) An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0052] (5) An single chain antibody ("SCA") is a genetically
engineered single chain molecule containing the variable region of
a light chain and the variable region of a heavy chain, linked by a
suitable, flexible polypeptide linker.
[0053] A molecule that has a "chimeric property" is a molecule that
is: 1) in part homologous and in part heterologous to a first
reference molecule; while 2) at the same time being in part
homologous and in part heterologous to a second reference molecule;
without 3) precluding the possibility of being at the same time in
part homologous and in part heterologous to still one or more
additional reference molecules. In a non-limiting embodiment, a
chimeric molecule may be prepared by assemblying a reassortment of
partial molecular sequences. In a non-limiting aspect, a chimeric
polynucleotide molecule may be prepared by synthesizing the
chimeric polynucleotide using plurality of molecular templates,
such that the resultant chimeric polynucleotide has properties of a
plurality of templates.
[0054] The term "cognate" as used herein refers to a gene sequence
that is evolutionarily and functionally related between species.
For example, but not limitation, in the human genome the human CD4
gene is the cognate gene to the mouse 3d4 gene, since the sequences
and structures of these two genes indicate that they are highly
homologous and both genes encode a protein which functions in
signaling T cell activation through MHC class II-restricted antigen
recognition.
[0055] A "comparison window," as used herein, refers to a
conceptual segment of at least 20 contiguous nucleotide positions
wherein a polynucleotide sequence may be compared to a reference
sequence of at least 20 contiguous nucleotides and wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 percent or less
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(1981) Adv. Appl. Math. 2: 482 by the homology alignment algorithm
of Needlemen and Wuncsch J. Mol. Biol. 48: 443 (1970), by the
search of similarity method of Pearson and Lipman Proc. Natl. Acad.
Sci. (U.S.A 85: 2444 (1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group. 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected.
[0056] As used herein, the term "complementarity-determining
region" and "CDR" refer to the art-recognized term as exemplified
by the Kabat and Chothia CDR definitions also generally known as
supervariable regions or hypervariable loops (Chothia and Leks,
1987; Clothia et al., 1989; Kabat et al., 1987; and Tramontano et
al., 1990). Variable region domains typically comprise the
amino-terminal approximately 105-115 amino acids of a
naturally-occurring immunoglobulin chain (e.g., amino acids 1-110),
although variable domains somewhat shorter or longer are also
suitable for forming single-chain antibodies.
[0057] "Conservative amino acid substitutions" refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleuci- ne,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0058] The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence. In
contradistinction, the term "complementary to" is used herein to
mean that the complementary sequence is homologous to all or a
portion of a reference polynucleotide sequence. For illustration,
the nucleotide sequence "TATAC" corresponds to a reference "TATAC"
and is complementary to a reference sequence "GTATA."
[0059] The term "degrading effective" amount refers to the amount
of enzyme which is required to process at least 50% of the
substrate, as compared to substrate not contacted with the enzyme.
Preferably, at least 80% of the substrate is degraded.
[0060] As used herein, the term "defined sequence framework" refers
to a set of defined sequences that are selected on a non-random
basis, generally on the basis of experimental data or structural
data; for example, a defined sequence framework may comprise a set
of amino acid sequences that are predicted to form a B-sheet
structure or may comprise a leucine zipper heptad repeat motif, a
zinc-finger domain, among other variations. A "defined sequence
kernal" is a set of sequences which encompass a limited scope of
variability. Whereas (1) a completely random 10-mer sequence of the
20 conventional amino acids can be any of (20).sup.10 sequences,
and (2) a pseudorandom 10-mer sequence of the 20 conventional amino
acids can be any of (20).sup.10 sequences but will exhibit a bias
for certain residues at certain positions and/or overall, (3) a
defined sequence kernal is a subset of sequences if each residue
position was allowed to be any of the allowable 20 conventional
amino acids (and/or allowable unconventional amino/imino acids). A
defined sequence kernal generally comprises variant and invariant
residue positions and/or comprises variant residue positions which
can comprise a residue selected from a defined subset of amino acid
residues), and the like, either segmentally or over the entire
length of the individual selected library member sequence. Defined
sequence kernels can refer to either amino acid sequences or
polynucleotide sequences. Of illustration and not limitation, the
sequences (NNK).sub.10 and (NNM).sub.10, wherein N represents A, T,
G, or C; K represents G or T; and M represents A or C, are defined
sequence kernels.
[0061] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a gel to
isolate the desired fragment.
[0062] The term "DNA shuffling" is used herein to indicate
recombination between substantially homologous but non-identical
sequences, in some embodiments DNA shuffling may involve crossover
via non-homologous recombination, such as via cer/lox and/or
flp/frt systems and the like.
[0063] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, such as a phytase polypeptide,
to which the paratope of an antibody, such as an phytase-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics. As used herein "epitope" refers to that portion of
an antigen or other macromolecule capable of forming a binding
interaction that interacts with the variable region binding body of
an antibody. Typically, such binding interaction is manifested as
an intermolecular contact with one or more amino acid residues of a
CDR.
[0064] The terms "fragment", "derivative" and "analog" when
referring to a reference polypeptide comprise a polypeptide which
retains at least one biological function or activity that is at
least essentially same as that of the reference polypeptide.
Furthermore, the terms "fragment", "derivative" or "analog" are
exemplified by a "pro-form" molecule, such as a low activity
proprotein that can be modified by cleavage to produce a mature
enzyme with significantly higher activity.
[0065] A method is provided herein for producing from a template
polypeptide a set of progeny polypeptides in which a "full range of
single amino acid substitutions" is represented at each amino acid
position. As used herein, "full range of single amino acid
substitutions" is in reference to the naturally encoded 20
naturally encoded polypeptide-forming alpha-amino acids, as
described herein.
[0066] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0067] "Genetic instability", as used herein, refers to the natural
tendency of highly repetitive sequences to be lost through a
process of reductive events generally involving sequence
simplification through the loss of repeated sequences. Deletions
tend to involve the loss of one copy of a repeat and everything
between the repeats.
[0068] The term "heterologous" means that one single-stranded
nucleic acid sequence is unable to hybridize to another
single-stranded nucleic acid sequence or its complement. Thus areas
of heterology means that areas of polynucleotides or
polynucleotides have areas or regions within their sequence which
are unable to hybridize to another nucleic acid or polynucleotide.
Such regions or areas are for example areas of mutations.
[0069] The term "homologous" or "homeologous" means that one
single-stranded nucleic acid nucleic acid sequence may hybridize to
a complementary single-stranded nucleic acid sequence. The degree
of hybridization may depend on a number of factors including the
amount of identity between the sequences and the hybridization
conditions such as temperature and salt concentrations as discussed
later. Preferably the region of identity is greater than about 5
bp, more preferably the region of identity is greater than 10
bp.
[0070] An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, also called CDR's. The extent of the framework region and
CDR's have been precisely defined (see, "Sequences of Proteins of
Immunological Interest," Kabat et al., 1987). The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a specie. As used herein, a "human framework
region" is a framework region that is substantially identical
(about 85 or more, usually 90-95 or more) to the framework region
of a naturally occurring human immunoglobulin. the framework region
of an antibody, that is the combined framework regions of the
constituent light and heavy chains, serves to position and align
the CDR's. The CDR's are primarily responsible for binding to an
epitope of an antigen.
[0071] The benefits of this invention extend to "industrial
applications" (or industrial processes), which term is used to
include applications in commercial industry proper (or simply
industry) as well as non-commercial industrial applications (e.g.
biomedical research at a non-profit institution). Relevant
applications include those in areas of diagnosis, medicine,
agriculture, manufacturing, and academia.
[0072] The term "identical" or "identity" means that two nucleic
acid sequences have the same sequence or a complementary sequence.
Thus, "areas of identity" means that regions or areas of a
polynucleotide or the overall polynucleotide are identical or
complementary to areas of another polynucleotide or the
polynucleotide.
[0073] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or enzyme present in a living animal is not
isolated, but the same polynucleotide or enzyme, separated from
some or all of the coexisting materials in the natural system, is
isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or enzymes could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0074] By "isolated nucleic acid" is meant a nucleic acid, e.g., a
DNA or RNA molecule, that is not immediately contiguous with the 5'
and 3' flanking sequences with which it normally is immediately
contiguous when present in the naturally occurring genome of the
organism from which it is derived. The term thus describes, for
example, a nucleic acid that is incorporated into a vector, such as
a plasmid or viral vector; a nucleic acid that is incorporated into
the genome of a heterologous cell (or the genome of a homologous
cell, but at a site different from that at which it naturally
occurs); and a nucleic acid that exists as a separate molecule, e
g., a DNA fragment produced by PCR amplification or restriction
enzyme digestion, or an RNA molecule produced by in vitro
transcription. The term also describes a recombinant nucleic acid
that forms part of a hybrid gene encoding additional polypeptide
sequences that can be used, for example, in the production of a
fusion protein.
[0075] As used herein "ligand" refers to a molecule, such as a
random peptide or variable segment sequence, that is recognized by
a particular receptor. As one of skill in the art will recognize, a
molecule (or macromolecular complex) can be both a receptor and a
ligand. In general, the binding partner having a smaller molecular
weight is referred to as the ligand and the binding partner having
a greater molecular weight is referred to as a receptor.
[0076] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis
et al, 1982, p. 146). Unless otherwise provided, ligation may be
accomplished using known buffers and conditions with 10 units of T4
DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0077] As used herein, "linker" or "spacer" refers to a molecule or
group of molecules that connects two molecules, such as a DNA
binding protein and a random peptide, and serves to place-the two
molecules in a preferred configuration, e.g., so that the random
peptide can bind to a receptor with minimal steric hindrance from
the DNA binding protein.
[0078] As used herein, a "molecular property to be evolved"
includes reference to molecules comprised of a polynucleotide
sequence, molecules comprised of a polypeptide sequence, and
molecules comprised in part of a polynucleotide sequence and in
part of a polypeptide sequence. Particularly relevant--but by no
means limiting--examples of molecular properties to be evolved
include enzymatic activities at specified conditions, such as
related to temperature; salinity; pressure; pH; and concentration
of glycerol, DMSO, detergent, &/or any other molecular species
with which contact is made in a reaction environment. Additional
particularly relevant--but by no means limiting--examples of
molecular properties to be evolved include stabilities--e.g. the
amount of a residual molecular property that is present after a
specified exposure time to a specified environment, such as may be
encountered during storage.
[0079] The term "mutations" means changes in the sequence of a
wild-type nucleic acid sequence or changes in the sequence of a
peptide. Such mutations may be point mutations such as transitions
or transversions. The mutations may be deletions, insertions or
duplications.
[0080] As used herein, the degenerate "N,N,G/T" nucleotide sequence
represents 32 possible triplets, where "N" can be A, C, G or T.
[0081] The term "naturally-occurring" as used herein as applied to
the object refers to the fact that an object can be found in
nature. For example, a polypeptide or polynucleotide sequence that
is present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally occurring.
Generally, the term naturally occurring refers to an object as
present in a non-pathological (un-diseased) individual, such as
would be typical for the species.
[0082] As used herein, a "nucleic acid molecule" is comprised of at
least one base or one base pair, depending on whether it is
single-stranded or double-stranded, respectively. Furthermore, a
nucleic acid molecule may belong exclusively or chimerically to any
group of nucleotide-containing molecules, as exemplified by, but
not limited to, the following groups of nucleic acid molecules:
RNA, DNA, genomic nucleic acids, non-genomic nucleic acids,
naturally occurring and not naturally occurring nucleic acids, and
synthetic nucleic acids. This includes, by way of non-limiting
example, nucleic acids associated with any organelle, such as the
mitochondria, ribosomal RNA, and nucleic acid molecules comprised
chimerically of one or more components that are not naturally
occurring along with naturally occurring components.
[0083] Additionally, a "nucleic acid molecule" may contain in part
one or more non-nucleotide-based components as exemplified by, but
not limited to, amino acids and sugars. Thus, by way of example,
but not limitation, a ribozyme that is in part nucleotide-based and
in part protein-based is considered a "nucleic acid molecule".
[0084] In addition, by way of example, but not limitation, a
nucleic acid molecule that is labeled with a detectable moiety,
such as a radioactive or alternatively a non-radioactive label, is
likewise considered a "nucleic acid molecule".
[0085] The terms "nucleic acid sequence coding for" or a "DNA
coding sequence of" or a "nucleotide sequence encoding" a
particular enzyme--as well as other synonymous terms--refer to a
DNA sequence which is transcribed and translated into an enzyme
when placed under the control of appropriate regulatory sequences.
A "promotor sequence" is a DNA regulatory region capable of binding
RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. The promoter is part of
the DNA sequence. This sequence region has a start codon at its 3'
terminus. The promoter sequence does include the minimum number of
bases where elements necessary to initiate transcription at levels
detectable above background. However, after the RNA polymerase
binds the sequence and transcription is initiated at the start
codon (3' terminus with a promoter), transcription proceeds
downstream in the 3' direction. Within the promotor sequence will
be found a transcription initiation site (conveniently defined by
mapping with nuclease S1) as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0086] The terms "nucleic acid encoding an enzyme (protein)" or
"DNA encoding an enzyme (protein)" or "polynucleotide encoding an
enzyme (protein)" and other synonymous terms encompasses a
polynucleotide which includes only coding sequence for the enzyme
as well as a polynucleotide which includes additional coding and/or
non-coding sequence.
[0087] In one preferred embodiment, a "specific nucleic acid
molecule species" is defined by its chemical structure, as
exemplified by, but not limited to, its primary sequence. In
another preferred embodiment, a specific "nucleic acid molecule
species" is defined by a function of the nucleic acid species or by
a function of a product derived from the nucleic acid species.
Thus, by way of non-limiting example, a "specific nucleic acid
molecule species" may be defined by one or more activities or
properties attributable to it, including activities or properties
attributable its expressed product.
[0088] The instant definition of "assembling a working nucleic acid
sample into a nucleic acid library" includes the process of
incorporating a nucleic acid sample into a vector-based collection,
such as by ligation into a vector and transformation of a host. A
description of relevant vectors, hosts, and other reagents as well
as specific non-limiting examples thereof are provided hereinafter.
The instant definition of "assembling a working nucleic acid sample
into a nucleic acid library" also includes the process of
incorporating a nucleic acid sample into a non-vector-based
collection, such as by ligation to adaptors. Preferably the
adaptors can anneal to PCR primers to facilitate amplification by
PCR.
[0089] Accordingly, in a non-limiting embodiment, a "nucleic acid
library" is comprised of a vector-based collection of one or more
nucleic acid molecules. In another preferred embodiment a "nucleic
acid library" is comprised of a non-vector-based collection of
nucleic acid molecules. In yet another preferred embodiment a
"nucleic acid library" is comprised of a combined collection of
nucleic acid molecules that is in part vector-based and in part
non-vector-based. Preferably, the collection of molecules
comprising a library is searchable and separable according to
individual nucleic acid molecule species.
[0090] The present invention provides a "nucleic acid construct" or
alternatively a "nucleotide construct" or alternatively a "DNA
construct". The term "construct" is used herein to describe a
molecule, such as a polynucleotide (e.g., a phytase polynucleotide)
may optionally be chemically bonded to one or more additional
molecular moieties, such as a vector, or parts of a vector. In a
specific--but by no means limiting--aspect, a nucleotide construct
is exemplified by a DNA expression DNA expression constructs
suitable for the transformation of a host cell.
[0091] An "oligonucleotide" (or synonymously an "oligo") refers to
either a single stranded polydeoxynucleotide or two complementary
polydeoxynucleotide strands which may be chemically synthesized.
Such synthetic oligonucleotides may or may not have a 5' phosphate.
Those that do not will not ligate to another oligonucleotide
without adding a phosphate with an ATP in the presence of a kinase.
A synthetic oligonucleofide will ligate to a fragment that has not
been dephosphorylated. To achieve polymerase-based amplification
(such as with PCR), a "32-fold degenerate oligonucleotide that is
comprised of, in series, at least a first homologous sequence, a
degenerate N,N,G/T sequence, and a second homologous sequence" is
mentioned. As used in this context, "homologous" is in reference to
homology between the oligo and the parental polynucleotide that is
subjected to the polymerase-based amplification.
[0092] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the coding sequence.
Operably linked means that the DNA sequences being linked are
typically contiguous and, where necessary to join two protein
coding regions, contiguous and in reading frame.
[0093] A coding sequence is "operably linked to" another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences are ultimately processed
to produce the desired protein.
[0094] As used herein the term "physiological conditions" refers to
temperature, pH, ionic strength, viscosity, and like biochemical
parameters which are compatible with a viable organism, and/or
which typically exist intracellularly in a viable cultured yeast
cell or mammalian cell. For example, the intracellular conditions
in a yeast cell grown under typical laboratory culture conditions
are physiological conditions. Suitable in vitro reaction conditions
for in vitro transcription cocktails are generally physiological
conditions. In general, in vitro physiological conditions comprise
50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45.degree. C. and 0.001-10 mM
divalent cation (e.g., Mg.sup.++, Ca.sup.++); preferably about 150
mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include
0.01-1.0 percent nonspecific protein (e.g., BSA). A non-ionic
detergent (Tween, NP40, Triton X-100) can often be present, usually
at about 0.001 to 2%, typically 0.05-0.2% (v/v). Particular aqueous
conditions may be selected by the practitioner according to
conventional methods. For general guidance, the following buffered
aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris
HCl, pH 5-8, with optional addition of divalent cation(s) and/or
metal chelators and/or non-ionic detergents and/or membrane
fractions and/or anti-foam agents and/or scintillants.
[0095] The term "population" as used herein means a collection of
components such as polynucleotides, portions or polynucleotides or
proteins. A "mixed population: means a collection of components
which belong to the same family of nucleic acids or proteins (i.e.,
are related) but which differ in their sequence (i.e., are not
identical) and hence in their biological activity.
[0096] A molecule having a "pro-form" refers to a molecule that
undergoes any combination of one or more covalent and noncovalent
chemical modifications (e.g. glycosylation, proteolytic cleavage,
dimerization or oligomerization, temperature-induced or pH-induced
conformational change, association with a co-factor, etc.) en route
to attain a more mature molecular form having a property difference
(e.g. an increase in activity) in comparison with the reference
pro-form molecule. When two or more chemical modification (e.g. two
proteolytic cleavages, or a proteolytic cleavage and a
deglycosylation) can be distinguished en route to the production of
a mature molecule, the reference precursor molecule may be termed a
"pre-pro-form" molecule.
[0097] As used herein, the term "pseudorandom" refers to a set of
sequences that have limited variability, such that, for example,
the degree of residue variability at another position, but any
pseudorandom position is allowed some degree of residue variation,
however circumscribed.
[0098] "Quasi-repeated units", as used herein, refers to the
repeats to be re-assorted and are by definition not identical.
Indeed the method is proposed not only for practically identical
encoding units produced by mutagenesis of the identical starting
sequence, but also the reassortment of similar or related sequences
which may diverge significantly in some regions. Nevertheless, if
the sequences contain sufficient homologies to be reassorted by
this approach, they can be referred to as "quasi-repeated"
units.
[0099] As used herein "random peptide library" refers to a set of
polynucleotide sequences that encodes a set of random peptides, and
to the set of random peptides encoded by those polynucleotide
sequences, as well as the fusion proteins contain those random
peptides.
[0100] As used herein, "random peptide sequence" refers to an amino
acid sequence composed of two or more amino acid monomers and
constructed by a stochastic or random process. A random peptide can
include framework or scaffolding motifs, which may comprise
invariant sequences.
[0101] As used herein, "receptor" refers to a molecule that has an
affinity for a given ligand. Receptors can be naturally occurring
or synthetic molecules. Receptors can be employed in an unaltered
state or as aggregates with other species. Receptors can be
attached, covalently or non-covalently, to a binding member, either
directly or via a specific binding substance. Examples of receptors
include, but are not limited to, antibodies, including monoclonal
antibodies and antisera reactive with specific antigenic
determinants (such as on viruses, cells, or other materials), cell
membrane receptors, complex carbohydrates and glycoproteins,
enzymes, and hormone receptors.
[0102] "Recombinant" enzymes refer to enzymes produced by
recombinant DNA techniques, i.e., produced from cells transformed
by an exogenous DNA construct encoding the desired enzyme.
"Synthetic" enzymes are those prepared by chemical synthesis.
[0103] The term "related polynucleotides" means that regions or
areas of the polynucleotides are identical and regions or areas of
the polynucleotides are heterologous.
[0104] "Reductive reassortment", as used herein, refers to the
increase in molecular diversity that is accrued through deletion
(and/or insertion) events that are mediated by repeated
sequences.
[0105] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence," "comparison window," "sequence identity," "percentage of
sequence identity," and "substantial identity."
[0106] A "reference sequence" is a defined sequence used as a basis
for a sequence comparison; a reference sequence may be a subset of
a larger sequence, for example, as a segment of a full-length cDNA
or gene sequence given in a sequence listing, or may comprise a
complete cDNA or gene sequence. Generally, a reference sequence is
at least 20 nucleotides in length, frequently at least 25
nucleotides in length, and often at least 50 nucleotides in length.
Since two polynucleotides may each (1) comprise a sequence (i.e., a
portion of the complete polynucleotide sequence) that is similar
between the two polynucleotides and (2) may further comprise a
sequence that is divergent between the two polynucleotides,
sequence comparisons between two (or more) polynucleotides are
typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare
local regions of sequence similarity.
[0107] "Repetitive Index (RI)", as used herein, is the average
number of copies of the quasi-repeated units contained in the
cloning vector.
[0108] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over the window of comparison. The term "percentage of sequence
identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, U, or I) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. This "substantial identity", as used herein,
denotes a characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence having at least 80 percent
sequence identity, preferably at least 85 percent identity, often
90 to 95 percent sequence identity, and most commonly at least 99
percent sequence identity as compared to a reference sequence of a
comparison window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison.
[0109] As known in the art "similarity" between two enzymes is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one enzyme to the sequence of a second
enzyme. Similarity may be determined by procedures which are
well-known in the art, for example, a BLAST program (Basic Local
Alignment Search Tool at the National Center for Biological
Information).
[0110] As used herein, the term "single-chain antibody" refers to a
polypeptide comprising a V.sub.H domain and a V.sub.L domain in
polypeptide linkage, generally liked via a spacer peptide (e.g.,
(Gly-Gly-Gly-Gly-Ser.sub.x), and which may comprise additional
amino acid sequences at the amino- and/or carboxy-termini. For
example, a single-chain antibody may comprise a tether segment for
linking to the encoding polynucleotide. As an example, a scFv is a
single-chain antibody. Single-chain antibodies are generally
proteins consisting of one or more polypeptide segments of at least
10 contiguous amino substantially encoded by genes of the
immunoglobulin superfamily (e.g., see Williams and Barclay, 1989,
pp. 361-368, which is incorporated herein by reference), most
frequently encoded by a rodent, non-human primate, avian, porcine
bovine, ovine, goat, or human heavy chain or light chain gene
sequence. A functional single-chain antibody generally contains a
sufficient portion of an immunoglobulin superfamily gene product so
as to retain the property of binding to a specific target molecule,
typically a receptor or antigen (epitope).
[0111] The members of a pair of molecules (e.g., an
antibody-antigen pair or a nucleic acid pair) are said to
"specifically bind" to each other if they bind to each other with
greater affinity than to other, non-specific molecules. For
example, an antibody raised against an antigen to which it binds
more efficiently than to a non-specific protein can be described as
specifically binding to the antigen. (Similarly, a nucleic acid
probe can be described as specifically binding to a nucleic acid
target if it forms a specific duplex with the target by base
pairing interactions (see above).)
[0112] "Specific hybridization" is defined herein as the formation
of hybrids between a first polynucleotide and a second
polynucleotide (e.g., a polynucleotide having a distinct but
substantially identical sequence to the first polynucleotide),
wherein substantially unrelated polynucleotide sequences do not
form hybrids in the mixture.
[0113] The term "specific polynucleotide" means a polynucleotide
having certain end points and having a certain nucleic acid
sequence. Two polynucleotides wherein one polynucleotide has the
identical sequence as a portion of the second polynucleotide but
different ends comprises two different specific
polynucleotides.
[0114] "Stringent hybridization conditions" means hybridization
will occur only if there is at least 90% identity, preferably at
least 95% identity and most preferably at least 97% identity
between the sequences. See Sambrook et al, 1989, which is hereby
incorporated by reference in its entirety.
[0115] Also included in the invention are polypeptides having
sequences that are "substantially identical" to the sequence of a
phytase polypeptide, such as one of SEQ ID 1. A "substantially
identical" amino acid sequence is a sequence that differs from a
reference sequence only by conservative amino acid substitutions,
for example, substitutions of one amino acid for another of the
same class (e.g., substitution of one hydrophobic amino acid, such
as isoleucine, valine, leucine, or methionine, for another, or
substitution of one polar amino acid for another, such as
substitution of arginine for lysine, glutamic acid for aspartic
acid, or glutamine for asparagine).
[0116] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence or by one or
more non-conservative substitutions, deletions, or insertions,
particularly when such a substitution occurs at a site that is not
the active site the molecule, and provided that the polypeptide
essentially retains its behavioural properties. For example, one or
more amino acids can be deleted from a phytase polypeptide,
resulting in modification of the structure of the polypeptide,
without significantly altering its biological activity. For
example, amino- or carboxyl-terminal amino acids that are not
required for phytase biological activity can be removed. Such
modifications can result in the development of smaller active
phytase polypeptides.
[0117] The present invention provides a "substantially pure
enzyme". The term "substantially pure enzyme" is used herein to
describe a molecule, such as a polypeptide (e.g., a phytase
polypeptide, or a fragment thereof) that is substantially free of
other proteins, lipids, carbohydrates, nucleic acids, and other
biological materials with which it is naturally associated. For
example, a substantially pure molecule, such as a polypeptide, can
be at least 60%, by dry weight, the molecule of interest. The
purity of the polypeptides can be determined using standard methods
including, e.g., polyacrylamide gel electrophoresis (e.g.,
SDS-PAGE), column chromatography (e.g., high performance liquid
chromatography (HPLC)), and amino-terminal amino acid sequence
analysis.
[0118] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual macromolecular species in
the composition), and preferably substantially purified fraction is
a composition wherein the object species comprises at least about
50 percent (on a molar basis) of all macromolecular species
present. Generally, a substantially pure composition will comprise
more than about 80 to 90 percent of all macromolecular species
present in the composition. Most preferably, the object species is
purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods)
wherein the composition consists essentially of a single
macromolecular species. Solvent species, small molecules (<500
Daltons), and elemental ion species are not considered
macromolecular species.
[0119] As used herein, the term "variable segment" refers to a
portion of a nascent peptide which comprises a random,
pseudorandom, or defined kernal sequence. A variable segment"
refers to a portion of a nascent peptide which comprises a random
pseudorandom, or defined kernal sequence. A variable segment can
comprise both variant and invariant residue positions, and the
degree of residue variation at a variant residue position may be
limited: both options are selected at the discretion of the
practitioner. Typically, variable segments are about 5 to 20 amino
acid residues in length (e.g., 8 to 10), although variable segments
may be longer and may comprise antibody portions or receptor
proteins, such as an antibody fragment, a nucleic acid binding
protein, a receptor protein, and the like.
[0120] The term "wild-type" means that the polynucleotide does not
comprise any mutations. A "wild type" protein means that the
protein will be active at a level of activity found in nature and
will comprise the amino acid sequence found in nature.
DETAILED DESCRIPTION OF THE INVENTION
[0121] The invention described herein is directed to the use of
repeated cycles of reductive reassortment, recombination and
selection which allow for the directed molecular evolution of
highly complex linear sequences, such as DNA, RNA or proteins
thorough recombination.
[0122] In vivo shuffling of molecules can be performed utilizing
the natural property of cells to recombine multimers. While
recombination in vivo has provided the major natural route to
molecular diversity, genetic recombination remains a relatively
complex process that involves 1) the recognition of homologies; 2)
strand cleavage, strand invasion, and metabolic steps leading to
the production of recombinant chiasma; and finally 3) the
resolution of chiasma into discrete recombined molecules. The
formation of the chiasma requires the recognition of homologous
sequences.
[0123] In a preferred embodiment, the invention relates to a method
for producing a hybrid polynucleotide from at least a first
polynucleotide and a second polynucleotide. The present invention
can be used to produce a hybrid polynucleotide by introducing at
least a first polynucleotide and a second polynucleotide which
share at least one region of partial sequence homology into a
suitable host cell. The regions of partial sequence homology
promote processes which result in sequence reorganization producing
a hybrid polynucleotide. The term "hybrid polynucleotide", as used
herein, is any nucleotide sequence which results from the method of
the present invention and contains sequence from at least two
original polynucleotide sequences. Such hybrid polynucleotides can
result from intermolecular recombination events which promote
sequence integration between DNA molecules. In addition, such
hybrid polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[0124] The invention provides a means for generating hybrid
polynucleotides which may encode biologically active hybrid
polypeptides. In one aspect, the original polynucleotides encode
biologically active polypeptides. The method of the invention
produces new hybrid polypeptides by utilizing cellular processes
which integrate the sequence of the original polynucleotides such
that the resulting hybrid polynucleotide encodes a polypeptide
demonstrating activities derived from the original biologically
active polypeptides. For example, the original polynucleotides may
encode a particular enzyme from different microorganisms. An enzyme
encoded by a first polynucleotide from one organism may, for
example, function effectively under a particular environmental
condition, e.g. high salinity. An enzyme encoded by a second
polynucleotide from a different organism may function effectively
under a different environmental condition, such as extremely high
temperatures. A hybrid polynucleotide containing sequences from the
first and second original polynucleotides may encode an enzyme
which exhibits characteristics of both enzymes encoded by the
original polynucleotides. Thus, the enzyme encoded by the hybrid
polynucleotide may function effectively under environmental
conditions shared by each of the enzymes encoded by the first and
second polynucleotides, e.g., high salinity and extreme
temperatures.
[0125] Enzymes encoded by the original polynucleotides of the
invention include, but are not limited to; oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases. A hybrid
polypeptide resulting from the method of the invention may exhibit
specialized enzyme activity not displayed in the original enzymes.
For example, following recombination and/or reductive reassortment
of polynucleotides encoding hydrolase activities, the resulting
hybrid polypeptide encoded by a hybrid polynucleotide can be
screened for specialized hydrolase activities obtained from each of
the original enzymes, i.e. the type of bond on which the hydrolase
acts and the temperature at which the hydrolase functions. Thus,
for example, the hydrolase may be screened to ascertain those
chemical functionalities which distinguish the hybrid hydrolase
from the original hydrolyases, such as: (a) amide (peptide bonds),
i.e. proteases; (b) ester bonds, i.e. esterases and lipases; (c)
acetals, i.e., glycosidases and, for example, the temperature, pH
or salt concentration at which the hybrid polypeptide
functions.
[0126] Sources of the original polynucleotides may be isolated from
individual organisms ("isolates"), collections of organisms that
have been grown in defined media ("enrichment cultures"), or, most
preferably, uncultivated organisms ("environmental samples"). The
use of a culture-independent approach to derive polynucleotides
encoding novel bioactivities from environmental samples is most
preferable since it allows one to access untapped resources of
biodiversity.
[0127] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample which may
be under-represented by several orders of magnitude compared to the
dominant species.
[0128] For example, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. Polynucleotides encoding activities of
interest are isolated from such libraries and introduced into a
host cell. The host cell is grown under conditions which promote
recombination and/or reductive reassortment creating potentially
active biomolecules with novel or enhanced activities.
[0129] The microorganisms from which the polynucleotide may be
prepared include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria, and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Polynucleotides may be isolated from
environmental samples in which case the nucleic acid may be
recovered without culturing of an organism or recovered from one or
more cultured organisms. In one aspect, such microorganisms may be
extremophiles, such as hyperthermophiles, psychrophiles,
psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms are particularly preferred. Such enzymes may
function at temperatures above 100.degree. C. in terrestrial hot
springs and deep sea thermal vents, at temperatures below 0.degree.
C. in arctic waters, in the saturated salt environment of the Dead
Sea, at pH values around 0 in coal deposits and geothermal
sulfur-rich springs, or at pH values greater than 11 in sewage
sludge. For example, several esterases and lipases cloned and
expressed from extremophilic organisms show high activity
throughout a wide range of temperatures and pHs.
[0130] Polynucleotides selected and isolated as hereinabove
described are introduced into a suitable host cell. A suitable host
cell is any cell which is capable of promoting recombination and/or
reductive reassortment. The selected polynucleotides are preferably
already in a vector which includes appropriate control sequences.
The host cell can be a higher eukaryotic cell, such as a mammalian
cell, or a lower eukaryotic cell, such as a yeast cell, or
preferably, the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation (Davis, Dibner, &
Battey, 1986).
[0131] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; and plant cells. The selection
of anappropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0132] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described by Gluzman, Cell, 23:175
(1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0133] Host cells containing the polynucleotides of interest can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying genes.
The culture conditions, such as temperature, pH and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan. The clones
which are identified as having the specified enzyme activity may
then be sequenced to identify the polynucleotide sequence encoding
an enzyme having the enhanced activity.
[0134] In another aspect, it is envisioned the method of the
present invention can be used to generate novel polynucleotides
encoding biochemical pathways from one or more operons or gene
clusters or portions thereof. For example, bacteria and many
eukaryotes have a coordinated mechanism for regulating genes whose
products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. Thus, a gene cluster
is a group of adjacent genes that are either identical or related,
usually as to their function. An example of a biochemical pathway
encoded by gene clusters are polyketides. Polyketides are molecules
which are an extremely rich source of bioactivities, including
antibiotics (such as tetracyclines and erythromycin), anti-cancer
agents (daunomycin), immunosuppressants (FK506 and rapamycin), and
veterinary products (monensin). Many polyketides (produced by
polyketide synthases) are valuable as therapeutic agents.
Polyketide synthases are multifunctional enzymes that catalyze the
biosynthesis of an enormous variety of carbon chains differing in
length and patterns of functionality and cyclization. Polyketide
synthase genes fall into gene clusters and at least one type
(designated type 1) of polyketide synthases have large size genes
and enzymes, complicating genetic manipulation and in vitro studies
of these genes/proteins.
[0135] The ability to select and combine desired components from a
library of polyketides, or fragments thereof, and postpolyketide
biosynthesis genes for generation of novel polyketides for study is
appealing. The method of the present invention makes it possible to
facilitate the production of novel polyketide synthases through
intermolecular recombination.
[0136] Preferably, gene cluster DNA can be isolated from different
organisms and ligated into vectors, particularly vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affect high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large DNA fragments,
such as gene clusters from mixed microbial samples. Once ligated
into an appropriate vector, two or more vectors containing
different polyketide synthase gene clusters can be introduced into
a suitable host cell. Regions of partial sequence homology shared
by the gene clusters will promote processes which result in
sequence reorganization resulting in a hybrid gene cluster. The
novel hybrid gene cluster can then be screened for enhanced
activities not found in the original gene clusters.
[0137] Therefore, in a preferred embodiment, the present invention
relates to a method for producing a biologically active hybrid
polypeptide and screening such a polypeptide for enhanced activity
by:
[0138] 1) introducing at least a first polynucleotide in operable
linkage and a second polynucleotide in operable linkage, said at
least first polynucleotide and second polynucleotide sharing at
least one region of partial sequence homology, into a suitable host
cell;
[0139] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage;
[0140] 3) expressing a hybrid polypeptide encoded by the hybrid
polynucleotide;
[0141] 4) screening the hybrid polypeptide under conditions which
promote identification of enhanced biological activity; and
[0142] 5) isolating the a polynucleotide encoding the hybrid
polypeptide.
[0143] Methods for screening for various enzyme activities are
known to those of skill in the art and discussed throughout the
present specification. Such methods may be employed when isolating
the polypeptides and polynucleotides of the present invention.
[0144] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g. vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus and yeast). Thus, for example, the DNA may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used as long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention.
[0145] A preferred type of vector for use in the present invention
contains an f-factor origin replication. The f-factor (or fertility
factor) in E. coli is a plasmid which effects high frequency
transfer of itself during conjugation and less frequent transfer of
the bacterial chromosome itself. A particularly preferred
embodiment is to use cloning vectors, referred to as "fosmids" or
bacterial artificial chromosome (BAC) vectors. These are derived
from E. coli f-factor which is able to stably integrate large
segments of genomic DNA. When integrated with DNA from a mixed
uncultured environmental sample, this makes it possible to achieve
large genomic fragments in the form of a stable "environmental DNA
library."
[0146] Another preferred type of vector for use in the present
invention is a cosmid vector. Cosmid vectors were originally
designed to clone and propagate large segments of genomic DNA.
Cloning into cosmid vectors is described in detail in Sambrook, et
al., Molecular Cloning A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, 1989.
[0147] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers.
[0148] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0149] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycol)tic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium.
[0150] The cloning strategy permits expression via both vector
driven and endogenous promoters; vector promotion may be important
with expression of genes whose endogenous promoter will not
function in E. coli.
[0151] The DNA isolated or derived from microorganisms can
preferably be inserted into a vector or a plasmid prior to probing
for selected DNA. Such vectors or plasmids are preferably those
containing expression regulatory sequences, including promoters,
enhancers and the like. Such polynucleotides can be part of a
vector and/or a composition and still be isolated, in that such
vector or composition is not part of its natural environment.
Particularly preferred phage or plasmid and methods for
introduction and packaging into them are described in detail in the
protocol set forth herein.
[0152] The selection of the cloning vector depends upon the
approach taken, for example, the vector can be any cloning vector
with an adequate capacity for multiply repeated copies of a
sequence, or multiple sequences that can be successfully
transformed and selected in a host cell. One example of such a
vector is described in "Polycos vectors: a system for packaging
filamentous phage and phagemid vectors using lambda phage packaging
extracts", Alting-Mecs M A, Short J M, Gene, 1993 Dec 27, 137:1,
93-100. Propagation/maintenance can be by an antibiotic resistance
carried by the cloning vector. After a period of growth, the
naturally abbreviated molecules are recovered and identified by
size fractionation on a gel or column, or amplified directly. The
cloning vector utilized may contain a selectable gene that is
disrupted by the insertion of the lengthy construct. As reductive
reassortment progresses, the number of repeated units is reduced
and the interrupted gene is again expressed and hence selection for
the processed construct can be applied. The vector may be an
expression/selection vector which will allow for the selection of
an expressed product possessing desirable biologically properties.
The insert may be positioned downstream of a functional promotor
and the desirable property screened by appropriate means.
[0153] In vivo reassortment is focused on "inter-molecular"
processes collectively referred to as "recombination" which in
bacteria, is generally viewed as a "RecA-dependent" phenomenon. The
present invention can rely on recombination processes of a host
cell to recombine and re-assort sequences, or the cells' ability to
mediate reductive processes to decrease the complexity of
quasi-repeated sequences in the cell by deletion. This process of
"reductive reassortment" occurs by an "intra-molecular",
RecA-independent process.
[0154] Therefore, in another aspect of the present invention, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs
containing consecutive sequences (original encoding sequences),
their insertion into an appropriate vector, and their subsequent
introduction into an appropriate host cell. The reassortment of the
individual molecular identities occurs by combinatorial processes
between the consecutive sequences in the construct possessing
regions of homology, or between quasi-repeated units. The
reassortment process recombines and/or reduces the complexity and
extent of the repeated sequences, and results in the production of
novel molecular species. Various treatments may be applied to
enhance the rate of reassortment. These could include treatment
with ultra-violet light, or DNA damaging chemicals, and/or the use
of host cell lines displaying enhanced levels of "genetic
instability". Thus the reassortment process may involve homologous
recombination or the natural property of quasi-repeated sequences
to direct their own evolution.
[0155] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In the present invention, "quasi-repeats" are
repeats that are not restricted to their original unit structure.
Quasi-repeated units can be presented as an array of sequences in a
construct; consecutive units of similar sequences. Once ligated,
the junctions between the consecutive sequences become essentially
invisible and the quasi-repetitive nature of the resulting
construct is now continuous at the molecular level. The deletion
process the cell performs to reduce the complexity of the resulting
construct operates between the quasi-repeated sequences. The
quasi-repeated units provide a practically limitless repertoire of
templates upon which slippage events can occur. The constructs
containing the quasi-repeats thus effectively provide sufficient
molecular elasticity that deletion (and potentially insertion)
events can occur virtually anywhere within the quasi-repetitive
units.
[0156] When the quasi-repeated sequences are all ligated in the
same orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, it is preferable with the present method that the sequences
are in the same orientation. Random orientation of quasi-repeated
sequences will result in the loss of reassortment efficiency, while
consistent orientation of the sequences will offer the highest
efficiency. However, while having fewer of the contiguous sequences
in the same orientation decreases the efficiency, it may still
provide sufficient elasticity for the effective recovery of novel
molecules. Constructs can be made with the quasi-repeated sequences
in the same orientation to allow higher efficiency.
[0157] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following:
[0158] a) Primers that include a poly-A head and poly-T tail which
when made single-stranded would provide orientation can be
utilized. This is accomplished by having the first few bases of the
primers made from RNA and hence easily removed RNAseH.
[0159] b) Primers that include unique restriction cleavage sites
can be utilized. Multiple sites, a battery of unique sequences, and
repeated synthesis and ligation steps would be required.
[0160] c) The inner few bases of the primer could be thiolated and
an exonuclease used to produce properly tailed molecules.
[0161] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced RI. The
re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The
recovery of cloning vectors with reduced RI can be effected by:
[0162] 1) The use of vectors only stably maintained when the
construct is reduced in complexity.
[0163] 2) The physical recovery of shortened vectors by physical
procedures. In this case, the cloning vector would be recovered
using standard plasmid isolation procedures and size fractionated
on either an agarose gel, or column with a low molecular weight cut
off utilizing standard procedures.
[0164] 3) The recovery of vectors containing interrupted genes
which can be selected when insert size decreases.
[0165] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0166] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, while the examples illustrated below demonstrate the
reassortment of nearly identical original encoding sequences
(quasi-repeats), this process is not limited to such nearly
identical repeats.
[0167] The following example demonstrates the method of the
invention. Encoding nucleic acid sequences (quasi-repeats) derived
from three (3) unique species are depicted. Each sequence encodes a
protein with a distinct set of properties. Each of the sequences
differs by a single or a few base pairs at a unique position in the
sequence which are designated "A", "B" and "C". The quasi-repeated
sequences are separately or collectively amplified and ligated into
random assemblies such that all possible permutations and
combinations are available in the population of ligated molecules.
The number of quasi-repeat units can be controlled by the assembly
conditions. The average number of quasi-repeated units in a
construct is defined as the repetitive index (RI).
[0168] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector, and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassortment
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in RI is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[0169] Optionally, the method comprises the additional step of
screening the library members of the shuffled pool to identify
individual shuffled library members having the ability to bind or
otherwise interact (e.g., such as catalytic antibodies) with a
predetermined macromolecule, such as for example a proteinaceous
receptor, peptide oligosaccharide, viron, or other predetermined
compound or structure.
[0170] The displayed polypeptides, antibodies, peptidomimetic
antibodies, and variable region sequences that are identified from
such libraries can be used for therapeutic, diagnostic, research
and related purposes (e.g., catalysts, solutes for increasing
osmolarity of an aqueous solution, and the like), and/or can be
subjected to one or more additional cycles of shuffling and/or
affinity selection. The method can be modified such that the step
of selecting for a phenotypic characteristic can be other than of
binding affinity for a predetermined molecule (e.g., for catalytic
activity, stability oxidation resistance, drug resistance, or
detectable phenotype conferred upon a host cell).
[0171] The present invention provides a method for generating
libraries of displayed antibodies suitable for affinity
interactions screening. The method comprises (1) obtaining first a
plurality of selected library members comprising a displayed
antibody and an associated polynucleotide encoding said displayed
antibody, and obtaining said associated polynucleotide encoding for
said displayed antibody and obtaining said associated
polynucleotides or copies thereof, wherein said associated
polynucleotides comprise a region of substantially identical
variable region framework sequence, and (2) introducing said
polynucleotides into a suitable host cell and growing the cells
under conditions which promote recombination and reductive
reassortment resulting in shuffled polynucleotides. CDR
combinations comprised by the shuffled pool are not present in the
first plurality of selected library members, said shuffled pool
composing a library of displayed antibodies comprising CDR
permutations and suitable for affinity interaction screening.
Optionally, the shuffled pool is subjected to affinity screening to
select shuffled library members which bind to a predetermined
epitope (antigen) and thereby selecting a plurality of selected
shuffled library members. Further, the plurality of selectively
shuffled library members can be shuffled and screened iteratively,
from 1 to about 1000 cycles or as desired until library members
having a desired binding affinity are obtained.
[0172] In another aspect of the invention, it is envisioned that
prior to or during recombination or reassortment, polynucleotides
generated by the method of the present invention can be subjected
to agents or processes which promote the introduction of mutations
into the original polynucleotides. The introduction of such
mutations would increase the diversity of resulting hybrid
polynucleotides and polypeptides encoded therefrom. The agents or
processes which promote mutagenesis can include, but are not
limited to: (+)-CC-1065, or a synthetic analog such as
(+)-CC-1065-(N3-Adenine), (see. Biochem. 31, 2822-2829 (1992)); a
N-acelylated or deacetylated 4'-fluro-4-aminobiphenyl adduct
capable of inhibiting DNA synthesis (see, for example,
Carcinogenesis vol. 13, No. 5, 751-758 (1992); or a N-acetylated or
deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA
synthesis (see also, Id. 751-758); trivalent chromium, a trivalent
chromium salt, a polycyclic aromatic hydrocarbon ("PAH") DNA adduct
capable of inhibiting DNA replication, such as
7-bromomethyl-benz[a]anthracene ("BMA"), tris(2,3-dibromopropyl)p-
hosphate ("Tris-BP"), 1,2-dibromo-3-chloropropane ("DBCP"),
2-bromoacrolein (2BA), benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide
("BPDE"), a platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[- 4,5-f]-quinoline
("N-hydroxy-IQ"), and N-hydroxy-2-amino-1-methyl-6-phenyl-
imidazo[4,5-f]-pyridine ("N-hydroxy-PhIP"). Especially preferred
"means for slowing or halting PCR amplification consist of UV light
(+)-CC-1065 and (+)-CC-1065-(N3-Adenine). Particularly encompassed
means are DNA adducts or polynucleotides comprising the DNA adducts
from the polynucleotides or polynucleotides pool, which can be
released or removed by a process including heating the solution
comprising the polynucleotides prior to further processing.
[0173] In another aspect the present invention is directed to a
method of producing recombinant proteins having biological activity
by treating a sample comprising double-stranded template
polynucleotides encoding a wild-type protein under conditions
according to the present invention which provide for the production
of hybrid or re-assorted polynucleotides.
[0174] The invention also provides the use of polynucleotide
shuffling to shuffle a population of viral genes (e.g., capsid
proteins, spike glycoproteins, polymerases, and proteases) or viral
genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses,
retroviruses, reoviruses and rhinoviruses). In an embodiment, the
invention provides a method for shuffling sequences encoding all or
portions of immunogenic viral proteins to generate novel
combinations of epitopes as well as novel epitopes created by
recombination; such shuffled viral proteins may comprise epitopes
or combinations of epitopes as well as novel epitopes created by
recombination; such shuffled viral proteins may comprise epitopes
or combinations of epitopes which are likely to arise in the
natural environment as a consequence of viral evolution; (e.g.,
such as recombination of influenza virus strains).
[0175] The invention also provides a method suitable for shuffling
polynucleotide sequences for generating gene therapy vectors and
replication-defective gene therapy constructs, such as may be used
for human gene therapy, including but not limited to vaccination
vectors for DNA-based vaccination, as well as anti-neoplastic gene
therapy and other general therapy formats.
[0176] In the polypeptide notation used herein, the left-hand
direction is the amino terminal direction and the right-hand
direction is the carboxy-terminal direction, in accordance with
standard usage and convention. Similarly, unless specified
otherwise, the left-hand end of single-stranded polynucleotide
sequences is the 5' end; the left-hand direction of double-stranded
polynucleotide sequences is referred to as the 5' direction. The
direction of 5' to 3' addition of nascent RNA transcripts is
referred to as the transcription direction; sequence regions on the
DNA strand having the same sequence as the RNA and which are 5' to
the 5' end of the RNA transcript are referred to as "upstream
sequences"; sequence regions on the DNA strand having the same
sequence as the RNA and which are 3' to the 3' end of the coding
RNA transcript are referred to as "downstream sequences".
[0177] Methodology
[0178] Nucleic acid shuffling is a method for in vitro or in vivo
homologous recombination of pools of shorter or smaller
polynucleotides to produce a polynucleotide or polynucleotides.
Mixtures of related nucleic acid sequences or polynucleotides are
subjected to sexual PCR to provide random polynucleotides, and
reassembled to yield a library or mixed population of recombinant
hybrid nucleic acid molecules or polynucleotides.
[0179] In contrast to cassette mutagenesis, only shuffling and
error-prone PCR allow one to mutate a pool of sequences blindly
(without sequence information other than primers).
[0180] The advantage of the mutagenic shuffling of this invention
over error-prone PCR alone for repeated selection can best be
explained with an example from antibody engineering. Consider DNA
shuffling as compared with error-prone PCR (not sexual PCR). The
initial library of selected pooled sequences can consist of related
sequences of diverse origin (i. e. antibodies from naive mRNA) or
can be derived by any type of mutagenesis (including shuffling) of
a single antibody gene. A collection of selected complementarity
determining regions ("CDRs") is obtained after the first round of
affinity selection. In the diagram the thick CDRs confer onto the
antibody molecule increased affinity for the antigen. Shuffling
allows the free combinatorial association of all of the CDR1s with
all of the CDR2s with all of the CDR3s, for example.
[0181] This method differs from error-prone PCR, in that it is an
inverse chain reaction. In error-prone PCR, the number of
polymerase start sites and the number of molecules grows
exponentially. However, the sequence of the polymerase start sites
and the sequence of the molecules remains essentially the same. In
contrast, in nucleic acid reassembly or shuffling of random
polynucleotides the number of start sites and the number (but not
size) of the random polynucleotides decreases over time. For
polynucleotides derived from whole plasmids the theoretical
endpoint is a single, large concatemeric molecule.
[0182] Since cross-overs occur at regions of homology,
recombination will primarily occur between members of the same
sequence family. This discourages combinations of CDRs that are
grossly incompatible (e.g., directed against different epitopes of
the same antigen). It is contemplated that multiple families of
sequences can be shuffled in the same reaction. Further, shuffling
generally conserves the relative order, such that, for example,
CDR1 will not be found in the position of CDR2.
[0183] Rare shufflants will contain a large number of the best (eg.
highest affinity) CDRs and these rare shufflants may be selected
based on their superior affinity.
[0184] CDRs from a pool of 100 different selected antibody
sequences can be permutated in up to 1006 different ways. This
large number of permutations cannot be represented in a single
library of DNA sequences. Accordingly, it is contemplated that
multiple cycles of DNA shuffling and selection may be required
depending on the length of the sequence and the sequence diversity
desired.
[0185] Error-prone PCR, in contrast, keeps all the selected CDRs in
the same relative sequence, generating a much smaller mutant
cloud.
[0186] The template polynucleotide which may be used in the methods
of this invention may be DNA or RNA. It may be of various lengths
depending on the size of the gene or shorter or smaller
polynucleotide to be recombined or reassembled. Preferably, the
template polynucleotide is from 50 bp to 50 kb. It is contemplated
that entire vectors containing the nucleic acid encoding the
protein of interest can be used in the methods of this invention,
and in fact have been successfully used.
[0187] The template polynucleotide may be obtained by amplification
using the PCR reaction (U.S. Pat. No. 4,683,202 and U.S. Pat. No.
4,683,195) or other amplification or cloning methods. However, the
removal of free primers from the PCR products before subjecting
them to pooling of the PCR products and sexual PCR may provide more
efficient results. Failure to adequately remove the primers from
the original pool before sexual PCR can lead to a low frequency of
crossover clones.
[0188] The template polynucleotide often should be double-stranded.
A double-stranded nucleic acid molecule is recommended to ensure
that regions of the resulting single-stranded polynucleotides are
complementary to each other and thus can hybridize to form a
double-stranded molecule.
[0189] It is contemplated that single-stranded or double-stranded
nucleic acid polynucleotides having regions of identity to the
template polynucleotide and regions of heterology to the template
polynucleotide may be added to the template polynucleotide, at this
step. It is also contemplated that two different but related
polynucleotide templates can be mixed at this step.
[0190] The double-stranded polynucleotide template and any added
double-or single-stranded polynucleotides are subjected to sexual
PCR which includes slowing or halting to provide a mixture of from
about 5 bp to 5 kb or more. Preferably the size of the random
polynucleotides is from about 10 bp to 1000 bp, more preferably the
size of the polynucleotides is from about 20 bp to 500 bp.
[0191] Alternatively, it is also contemplated that double-stranded
nucleic acid having multiple nicks may be used in the methods of
this invention. A nick is a break in one strand of the
double-stranded nucleic acid. The distance between such nicks is
preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.
This can provide areas of self-priming to produce shorter or
smaller polynucleotides to be included with the polynucleotides
resulting from random primers, for example.
[0192] The concentration of any one specific polynucleotide will
not be greater than 1% by weight of the total polynucleotides, more
preferably the concentration of any one specific nucleic acid
sequence will not be greater than 0.1% by weight of the total
nucleic acid.
[0193] The number of different specific polynucletides in the
mixture will be at least about 100, preferably at least about 500,
and more preferably at least about 1000.
[0194] At this step single-stranded or double-stranded
polynucleotides, either synthetic or natural, may be added to the
random double-stranded shorter or smaller polynucleotides in order
to increase the heterogeneity of the mixture of
polynucleotides.
[0195] It is also contemplated that populations of double-stranded
randomly broken polynucleotides may be mixed or combined at this
step with the polynucleotides from the sexual PCR process and
optionally subjected to one or more additional sexual PCR
cycles.
[0196] Where insertion of mutations into the template
polynucleotide is desired, single-stranded or double-stranded
polynucleotides having a region of identity to the template
polynucleotide and a region of heterology to the template
polynucleotide maybe added in a 20 fold excess by weight as
compared to the total nucleic acid, more preferably the
single-stranded polynucleotides may be added in a 10 fold excess by
weight as compared to the total nucleic acid.
[0197] Where a mixture of different but related template
polynucleotides is desired, populations of polynucleotides from
each of the templates may be combined at a ratio of less than about
1:100, more preferably the ratio is less than about 1:40. For
example, a backcross of the wild-type polynucleotide with a
population of mutated polynucleotide may be desired to eliminate
neutral mutations (e.g., mutations yielding an insubstantial
alteration in the phenotypic property being selected for). In such
an example, the ratio of randomly provided wild-type
polynucleotides which may be added to the randomly provided sexual
PCR cycle hybrid polynucleotides is approximately 1:1 to about
100:1, and more preferably from 1:1 to 40:1.
[0198] The mixed population of random polynucleotides are denatured
to form single-stranded polynucleotides and then re-annealed. Only
those single-stranded polynucleotides having regions of homology
with other single-stranded polynucleotides will re-anneal.
[0199] The random polynucleotides may be denatured by heating. One
skilled in the art could determine the conditions necessary to
completely denature the double-stranded nucleic acid. Preferably
the temperature is from 80.degree. C. to 100.degree. C., more
preferably the temperature is from 90.degree. C. to 96.degree. C.
other methods which may be used to denature the polynucleotides
include pressure (36) and pH.
[0200] The polynucleotides may be re-annealed by cooling.
Preferably the temperature is from 20.degree. C. to 75.degree. C.,
more preferably the temperature is from 40.degree. C. to 65.degree.
C. If a high frequency of crossovers is needed based on an average
of only 4 consecutive bases of homology, recombination can be
forced by using a low annealing temperature, although the process
becomes more difficult. The degree of renaturation which occurs
will depend on the degree of homology between the population of
single-stranded polynucleotides.
[0201] Renaturation can be accelerated by the addition of
polyethylene glycol ("PEG") or salt. The salt concentration is
preferably from 0 mM to 200 mM, more preferably the salt
concentration is from 10 mM to 100 mm. The salt may be KCl or NaCl.
The concentration of PEG is preferably from 0% to 20%, more
preferably from 5% to 10%.
[0202] The annealed polynucleotides are next incubated in the
presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP,
DGTP and dTTP). The nucleic acid polymerase may be the Klenow
fragment, the Taq polymerase or any other DNA polymerase known in
the art
[0203] The approach to be used for the assembly depends on the
minimum degree of homology that should still yield crossovers. If
the areas of identity are large, Taq polymerase can be used with an
annealing temperature of between 45-65.degree. C. If the areas of
identity are small, Klenow polymerase can be used with an annealing
temperature of between 20-30.degree. C. One skilled in the art
could vary the temperature of annealing to increase the number of
cross-overs achieved.
[0204] The polymerase may be added to the random polynucleotides
prior to annealing, simultaneously with annealing or after
annealing.
[0205] The cycle of denaturation, renaturation and incubation in
the presence of polymerase is referred to herein as shuffling or
reassembly of the nucleic acid. This cycle is repeated for a
desired number of times. Preferably the cycle is repeated from 2 to
50 times, more preferably the sequence is repeated from 10 to 40
times.
[0206] The resulting nucleic acid is a larger double-stranded
polynucleotide of from about 50 bp to about 100 kb, preferably the
larger polynucleotide is from 500 bp to 50 kb.
[0207] This larger polynucleotides may contain a number of copies
of a polynucleotide having the same size as the template
polynucleotide in tandem. This concatemeric polynucleotide is then
denatured into single copies of the template polynucleotide. The
result will be a population of polynucleotides of approximately the
same size as the template polynucleotide. The population will be a
mixed population where single or double-stranded polynucleotides
having an area of identity and an area of heterology have been
added to the template polynucleotide prior to shuffling.
[0208] These polynucleotides are then cloned into the appropriate
vector and the ligation mixture used to transform bacteria.
[0209] It is contemplated that the single polynucleotides may be
obtained from the larger concatemeric polynucleotide by
amplification of the single polynucleotide prior to cloning by a
variety of methods including PCR (U.S. Pat. No. 4,683,195 and U.S.
Pat. No. 4,683,202), rather than by digestion of the
concatemer.
[0210] The vector used for cloning is not critical provided that it
will accept a polynucleotide of the desired size. If expression of
the particular polynucleotide is desired, the cloning vehicle
should further comprise transcription and translation signals next
to the site of insertion of the polynucleotide to allow expression
of the polynucleotide in the host cell. Preferred vectors include
the pUC series and the pBR series of plasmids.
[0211] The resulting bacterial population will include a number of
recombinant polynucleotides having random mutations. This mixed
population may be tested to identify the desired recombinant
polynucleotides. The method of selection will depend on the
polynucleotide desired.
[0212] For example, if a polynucleotide which encodes a protein
with increased binding efficiency to a ligand is desired, the
proteins expressed by each of the portions of the polynucleotides
in the population or library may be tested for their ability to
bind to the ligand by methods known in the art (i.e. panning,
affinity chromatography). If a polynucleotide which encodes for a
protein with increased drug resistance is desired, the proteins
expressed by each of the polynucleotides in the population or
library may be tested for their ability to confer drug resistance
to the host organism. One skilled in the art, given knowledge of
the desired protein, could readily test the population to identify
polynucleotides which confer the desired properties onto the
protein.
[0213] It is contemplated that one skilled in the art could use a
phage display system in which fragments of the protein are
expressed as fusion proteins on the phage surface (Pharmacia,
Milwaukee Wis.). The recombinant DNA molecules are cloned into the
phage DNA at a site which results in the transcription of a fusion
protein a portion of which is encoded by the recombinant DNA
molecule. The phage containing the recombinant nucleic acid
molecule undergoes replication and transcription in the cell. The
leader sequence of the fusion protein directs the transport of the
fusion protein to the tip of the phage particle. Thus the fusion
protein which is partially encoded by the recombinant DNA molecule
is displayed on the phage particle for detection and selection by
the methods described above.
[0214] It is further contemplated that a number of cycles of
nucleic acid shuffling may be conducted with polynucleotides from a
sub-population of the first population, which sub-population
contains DNA encoding the desired recombinant protein. In this
manner, proteins with even higher binding affinities or enzymatic
activity could be achieved.
[0215] It is also contemplated that a number of cycles of nucleic
acid shuffling may be conducted with a mixture of wild-type
polynucleotides and a sub-population of nucleic acid from the first
or subsequent rounds of nucleic acid shuffling in order to remove
any silent mutations from the sub-population.
[0216] Any source of nucleic acid, in purified form can be utilized
as the starting nucleic acid. Thus the process may employ DNA or
RNA including messenger RNA, which DNA or RNA may be single or
double stranded. In addition, a DNA-RNA hybrid which contains one
strand of each may be utilized. The nucleic acid sequence may be of
various lengths depending on the size of the nucleic acid sequence
to be mutated. Preferably the specific,nucleic acid sequence is
from 50 to 50000 base pairs. It is contemplated that entire vectors
containing the nucleic acid encoding the protein of interest may be
used in the methods of this invention.
[0217] The nucleic acid may be obtained from any source, for
example, from plasmids such a pBR322, from cloned DNA or RNA or
from natural DNA or RNA from any source including bacteria, yeast,
viruses and higher organisms such as plants or animals. DNA or RNA
may be extracted from blood or tissue material. The template
polynucleotide may be obtained by amplification using the
polynucleotide chain reaction (PCR) (U.S. Pat. No. 4,683,202 and
U.S. Pat. No. 4,683,195). Alternatively, the polynucleotide may be
present in a vector present in a cell and sufficient nucleic acid
may be obtained by culturing the cell and extracting the nucleic
acid from the cell by methods known in the art.
[0218] Any specific nucleic acid sequence can be used to produce
the population of hybrids by the present process. It is only
necessary that a small population of hybrid sequences of the
specific nucleic acid sequence exist or be created prior to the
present process.
[0219] The initial small population of the specific nucleic acid
sequences having mutations may be created by a number of different
methods. Mutations may be created by error-prone PCR. Error-prone
PCR uses low-fidelity polymerization conditions to introduce a low
level of point mutations randomly over a long sequence.
Alternatively, mutations can be introduced into the template
polynucleotide by oligonucleotide-directed mutagenesis. In
oligonucleotide-directed mutagenesis, a short sequence of the
polynucleotide is removed from the polynucleotide using restriction
enzyme digestion and is replaced with a synthetic polynucleotide in
which various bases have been altered from the original sequence.
The polynucleotide sequence can also be altered by chemical
mutagenesis. Chemical mutagens include, for example, sodium
bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
other agents which are analogues of nucleotide precursors include
nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.
Generally, these agents are added to the PCR reaction in place of
the nucleotide precursor thereby mutating the sequence.
Intercalating agents such as proflavine, acriflavine, quinacrine
and the like can also be used. Random mutagenesis of the
polynucleotide sequence can also be achieved by irradiation with
X-rays or ultraviolet light. Generally, plasmid polynucleotides so
mutagenized are introduced into E. coli and propagated as a pool or
library of hybrid plasmids.
[0220] Alternatively the small mixed population of specific nucleic
acids may be found in nature in that they may consist of different
alleles of the same gene or the same gene from different related
species (i.e., cognate genes). Alternatively, they may be related
DNA sequences found within one species, for example, the
immunoglobulin genes.
[0221] Once the mixed population of the specific nucleic acid
sequences is generated, the polynucleotides can be used directly or
inserted into an appropriate cloning vector, using techniques
well-known in, the art.
[0222] The choice of vector depends on the size of the
polynucleotide sequence and the host cell to be employed in the
methods of this invention. The templates of this invention may be
plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses,
parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and
the like), or selected portions thereof (e.g., coat protein, spike
glycoprotein, capsid protein). For example, cosmids and phagemids
are preferred where the specific nucleic acid sequence to be
mutated is larger because these vectors are able to stably
propagate large polynucleotides.
[0223] If the mixed population of the specific nucleic acid
sequence is cloned into a vector it can be clonally amplified by
inserting each vector into a host cell and allowing the host cell
to amplify the vector. This is referred to as clonal amplification
because while the absolute number of nucleic acid sequences
increases, the number of hybrids does not increase. Utility can be
readily determined by screening expressed polypeptides.
[0224] The DNA shuffling method of this invention can be performed
blindly on a pool of unknown sequences. By adding to the reassembly
mixture oligonucleotides (with ends that are homologous to the
sequences being reassembled) any sequence mixture can be
incorporated at any specific position into another sequence
mixture. Thus, it is contemplated that mixtures of synthetic
oligonucleotides, PCR polynucleotides or even whole genes can be
mixed into another sequence library at defined positions. The
insertion of one sequence (mixture) is independent from the
insertion of a sequence in another part of the template. Thus, the
degree of recombination, the homology required, and the diversity
of the library can be independently and simultaneously varied along
the length of the reassembled DNA.
[0225] This approach of mixing two genes may be useful for the
humanization of antibodies from murine hybridomas. The approach of
mixing two genes or inserting alternative sequences into genes may
be useful for any therapeutically used protein, for example,
interleukin I, antibodies, tPA and growth hormone. The approach may
also be useful in any nucleic acid for example, promoters or
introns or 31 untranslated region or 51 untranslated regions of
genes to increase expression or alter specificity of expression of
proteins. The approach may also be used to mutate ribozymes or
aptamers.
[0226] Shuffling requires the presence of homologous regions
separating regions of diversity. Scaffold-like protein structures
may be particularly suitable for shuffling. The conserved scaffold
determines the overall folding by self-association, while
displaying relatively unrestricted loops that mediate the specific
binding. Examples of such scaffolds are the immunoglobulin
beta-barrel, and the four-helix bundle which are well-known in the
art. This shuffling can be used to create scaffold-like proteins
with various combinations of mutated sequences for binding.
[0227] Saturation Mutagenesis
[0228] In one aspect, this invention provides for the use of
proprietary codon primers (containing a degenerate N,N,G/T
sequence) to introduce point mutations into a polynucleotide, so as
to generate a set of progeny polypeptides in which a full range of
single amino acid substitutions is represented at each amino acid
position. The oligos used are comprised contiguously of a first
homologous sequence, a degenerate N,N,G/T sequence, and preferably
but not necessarily a second homologous sequence. The downstream
progeny translational products from the use of such oligos include
all possible amino acid changes at each amino acid site along the
polypeptide, because the degeneracy of the N,N,G/T sequence
includes codons for all 20 amino acids.
[0229] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,G/T cassette) is used for subjecting each original
codon in a parental polynucleotide template to a full range of
codon substitutions. In another aspect, at least two degenerate
N,N,G/T cassettes are used--either in the same oligo or not, for
subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions.
Thus, more than one N,N,G/T sequence can be contained in one oligo
to introduce amino acid mutations at more than one site. This
plurality of N,N,G/T sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligos serviceable for introducing additions and
deletions can be used either alone or in combination with the
codons containing an N,N,G/T sequence, to introduce any combination
or permutation of amino acid additions, deletions, and/or
substitutions.
[0230] In a particular exemplification, it is possible to
simultaneously mutagenize two or more contiguous amino acid
positions using an oligo that contains contiguous N,N,G/T triplets,
i.e. a degenerate (N,N,G/T)n sequence.
[0231] In another aspect, the present invention provides for the
use of degenerate cassettes having less degeneracy than the N,N,G/T
sequence. For example, it may be desirable in some instances to use
(e.g. in an oligo) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two postitions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
[0232] It is appreciated, however, that the use of a degenerate
N,N,G/T triplet as disclosed in the instant invention is
advantageous for several reasons. In one aspect, this invention
provides a means to systematically and fairly easily generate the
substitution of the full range of possible amino acids (for a total
of 20 amino acids) into each and every amino acid position in a
polypeptide. Thus, for a 100 amino acid polypeptide, the instant
invention provides a way to systematically and fairly easily
generate 2000 distinct species (i.e. 20 possible amino acids per
position .times.100 amino acid positions). It is appreciated that
there is provided, through the use of an oligo containing a
degenerate N,N,G/T triplet, 32 individual sequences that code for
20 possible amino acids. Thus, in a reaction vessel in which a
parental polynucleotide sequence is subjected to saturation
mutagenesis using one such oligo, there are generated 32 distinct
progeny polynucleotides encoding 20 distinct polypeptides. In
contrast, the use of a non-degenerate oligo in site-directed
mutagenesis leads to only one progeny polypeptide product per
reaction vessel.
[0233] Thus, in a preferred embodiment of this invention, each
saturation mutagenesis reaction vessel contains polynucleotides
encoding at least 20 progeny polypeptide molecules such that all 20
amino acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide. The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g. cloned into a suitable E.
coli host using an expression vector) and subjected to expression
screening. When an individual progeny polypeptide is identified by
screening to display a favorable change in property (when compared
to the parental polypeptide), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[0234] It is appreciated that upon mutagenizing each and every
amino acid position in a parental polypeptide using saturation
mutagenesis as disclosed herein, favorable amino acid changes may
be identified at more than one amino acid position. One or more new
progeny molecules can be generated that contain a combination of
all or part of these favorable amino acid substitutions. For
example, if 2 specific favorable amino acid changes are identified
in each of 3 amino acid positions in a polypeptide, the
permutations include 3 possibilities at each position (no change
from the original amino acid, and each of two favorable changes)
and 3 positions. Thus, there are 3.times.3.times.3 or 27 total
possibilities, including 7 that were previously examined--6 single
point mutations (i.e. 2 at each of three positions) and no change
at any position.
[0235] In yet another aspect, site-saturation mutagenesis can be
used together with shuffling, chimerization, recombination and
other mutagenizing processes, along with screening. This invention
provides for the use of any mutagenizing process(es), including
saturation mutagenesis, in an iterative manner. In one
exemplification, the iterative use of any mutagenizing process(es)
is used in combination with screening.
[0236] Thus, in a non-limiting exemplification, this invention
provides for the use of saturation mutagenesis in combination with
additional mutagenization processes, such as process where two or
more related polynucleotides are introduced into a suitable host
cell such that a hybrid polynucleotide is generated by
recombination and reductive reassortment.
[0237] In Vitro Shuffling
[0238] The equivalents of some standard genetic matings may also be
performed by shuffling in vitro. For example, a "molecular
backcross" can be performed by repeatedly mixing the hybrid's
nucleic acid with the wild-type nucleic acid while selecting for
the mutations of interest. As in traditional breeding, this
approach can be used to combine phenotypes from different sources
into a background of choice. It is useful, for example, for the
removal of neutral mutations that affect unselected characteristics
(i.e. immunogenicity). Thus it can be useful to determine which
mutations in a protein are involved in the enhanced biological
activity and which are not, an advantage which cannot be achieved
by error-prone mutagenesis or cassette mutagenesis methods.
[0239] Large, functional genes can be assembled correctly from a
mixture of small random polynucleotides. This reaction may be of
use for the reassembly of genes from the highly fragmented DNA of
fossils. In addition random nucleic acid fragments from fossils may
be combined with polynucleotides from similar genes from related
species.
[0240] It is also contemplated that the method of this invention
can be used for the in vitro amplification of a whole genome from a
single cell as is needed for a variety of research and diagnostic
applications. DNA amplification by PCR is in practice limited to a
length of about 40 kb. Amplification of a whole genome such as that
of E. coli (5, 000 kb) by PCR would require about 250 primers
yielding 125 forty kb polynucleotides. This approach is not
practical due to the unavailability of sufficient sequence data. On
the other hand, random production of polynucleotides of the genome
with sexual PCR cycles, followed by gel purification of small
polynucleotides will provide a multitude of possible primers. Use
of this mix of random small polynucleotides as primers in a PCR
reaction alone or with the whole genome as the template should
result in an inverse chain reaction with the theoretical endpoint
of a single concatemer containing many copies of the genome.
[0241] 100 fold amplification in the copy number and an average
polynucleotide size of greater than 50 kb may be obtained when only
random polynucleotides are used. It is thought that the larger
concatemer is generated by overlap of many smaller polynucleotides.
The quality of specific PCR products obtained using synthetic
primers will be indistinguishable from the product obtained from
unamplified DNA. It is expected that this approach will be useful
for the mapping of genomes.
[0242] The polynucleotide to be shuffled can be produced as random
or non-random polynucleotides, at the discretion of the
practitioner.
[0243] In Vivo Shuffling
[0244] In an embodiment of in vivo shuffling, the mixed population
of the specific nucleic acid sequence is introduced into bacterial
or eukaryotic cells under conditions such that at least two
different nucleic acid sequences are present in each host cell. The
polynucleotides can be introduced into the host cells by a variety
of different methods. The host cells can be transformed with the
smaller polynucleotides using methods known in the art, for example
treatment with calcium chloride. If the polynucleotides are
inserted into a phage genome, the host cell can be transfected with
the recombinant phage genome having the specific nucleic acid
sequences. Alternatively, the nucleic acid sequences can be
introduced into the host cell using electroporation, transfection,
lipofection, biolistics, conjugation, and the like.
[0245] In general, in this embodiment, the specific nucleic acids
sequences will be present in vectors which are capable of stably
replicating the sequence in the host cell. In addition, it is
contemplated that the vectors will encode a marker gene such that
host cells having the vector can be selected. This ensures that the
mutated specific nucleic acid sequence can be recovered after
introduction into the host cell. However, it is contemplated that
the entire mixed population of the specific nucleic acid sequences
need not be present on a vector sequence. Rather only a sufficient
number of sequences need be cloned into vectors to ensure that
after introduction of the polynucleotides into the host cells each
host cell contains one vector having at least one specific nucleic
acid sequence present therein. It is also contemplated that rather
than having a subset of the population of the specific nucleic
acids sequences cloned into vectors, this subset may be already
stably integrated into the host cell.
[0246] It has been found that when two polynucleotides which have
regions of identity are inserted into the host cells homologous
recombination occurs between the two polynucleotides. Such
recombination between the two mutated specific nucleic acid
sequences will result in the production of double or triple hybrids
in some situations.
[0247] It has also been found that the frequency of recombination
is increased if some of the mutated specific nucleic acid sequences
are present on linear nucleic acid molecules. Therefore, in a
preferred embodiment, some of the specific nucleic acid sequences
are present on linear polynucleotides.
[0248] After transformation, the host cell transformants are placed
under selection to identify those host cell transformants which
contain mutated specific nucleic acid sequences having the
qualities desired. For example, if increased resistance to a
particular drug is desired then the transformed host cells may be
subjected to increased concentrations of the particular drug and
those transformants producing mutated proteins able to confer
increased drug resistance will be selected. If the enhanced ability
of a particular protein to bind to a receptor is desired, then
expression of the protein can be induced from the transformants and
the resulting protein assayed in a ligand binding assay by methods
known in the art to identify that subset of the mutated population
which shows enhanced binding to the ligand. Alternatively, the
protein can be expressed in another system to ensure proper
processing.
[0249] Once a subset of the first recombined specific nucleic acid
sequences (daughter sequences) having the desired characteristics
are identified, they are then subject to a second round of
recombination.
[0250] In the second cycle of recombination, the recombined
specific nucleic acid sequences may be mixed with the original
mutated specific nucleic acid sequences (parent sequences) and the
cycle repeated as described above. In this way a set of second
recombined specific nucleic acids sequences can be identified which
have enhanced characteristics or encode for proteins having
enhanced properties. This cycle can be repeated a number of times
as desired.
[0251] It is also contemplated that in the second or subsequent
recombination cycle, a backcross can be performed. A molecular
backcross can be performed by mixing the desired specific nucleic
acid sequences with a large number of the wild-type sequence, such
that at least one wild-type nucleic acid sequence and a mutated
nucleic acid sequence are present in the same host cell after
transformation. Recombination with the wild-type specific nucleic
acid sequence will eliminate those neutral mutations that may
affect unselected characteristics such as immunogenicity but not
the selected characteristics.
[0252] In another embodiment of this invention, it is contemplated
that during the first round a subset of the specific nucleic acid
sequences can be generated as smaller polynucleotides by slowing or
halting their PCR amplification prior to introduction into the host
cell. The size of the polynucleotides must be large enough to
contain some regions of identity with the other sequences so as to
homologously recombine with the other sequences. The size of the
polynucleotides will range from 0.03 kb to 100 kb more preferably
from 0. 2 kb to 10 kb. It is also contemplated that in subsequent
rounds, all of the specific nucleic acid sequences other than the
sequences selected from the previous round may be utilized to
generate PCR polynucleotides prior to introduction into the host
cells.
[0253] The shorter polynucleotide sequences can be single-stranded
or double-stranded. If the sequences were originally
single-stranded and have become double-stranded they can be
denatured with heat, chemicals or enzymes prior to insertion into
the host cell. The reaction conditions suitable for separating the
strands of nucleic acid are well known in the art.
[0254] The steps of this process can be repeated indefinitely,
being limited only by the number of possible hybrids which can be
achieved. After a certain number of cycles, all possible hybrids
will have been achieved and further cycles are redundant.
[0255] In an embodiment the same mutated template nucleic acid is
repeatedly recombined and the resulting recombinants selected for
the desired characteristic.
[0256] Therefore, the initial pool or population of mutated
template nucleic acid is cloned into a vector capable of
replicating in a bacteria such as E. coli. The particular vector is
not essential, so long as it is capable of autonomous replication
in E. coli. In a preferred embodiment, the vector is designed to
allow the expression and production of any protein encoded by the
mutated specific nucleic acid linked to the vector. It is also
preferred that the vector contain a gene encoding for a selectable
marker.
[0257] The population of vectors containing the pool of mutated
nucleic acid sequences is introduced into the E. coli host cells.
The vector nucleic acid sequences may be introduced by
transformation, transfection or infection in the case of phage. The
concentration of vectors used to transform the bacteria is such
that a number of vectors is introduced into each cell. Once present
in the cell, the efficiency of homologous recombination is such
that homologous recombination occurs between the various vectors.
This results in the generation of hybrids (daughters) having a
combination of mutations which differ from the original parent
mutated sequences.
[0258] The host cells are then clonally replicated and selected for
the marker gene present on the vector. Only those cells having a
plasmid will grow under the selection.
[0259] The host cells which contain a vector are then tested for
the presence of favorable mutations. Such testing may consist of
placing the cells under selective pressure, for example, if the
gene to be selected is an improved drug resistance gene. If the
vector allows expression of the protein encoded by the mutated
nucleic acid sequence, then such selection may include allowing
expression of the protein so encoded, isolation of the protein and
testing of the protein to determine whether, for example, it binds
with increased efficiency to the ligand of interest.
[0260] Once a particular daughter mutated nucleic acid sequence has
been identified which confers the desired characteristics, the
nucleic acid is isolated either already linked to the vector or
separated from the vector. This nucleic acid is then mixed with the
first or parent population of nucleic acids and the cycle is
repeated.
[0261] It has been shown that by this method nucleic acid sequences
having enhanced desired properties can be selected.
[0262] In an alternate embodiment, the first generation of hybrids
are retained in the cells and the parental mutated sequences are
added again to the cells. Accordingly, the first cycle of
Embodiment I is conducted as described above. However, after the
daughter nucleic acid sequences are identified, the host cells
containing these sequences are retained.
[0263] The parent mutated specific nucleic acid population, either
as polynucleotides or cloned into the same vector is introduced
into the host cells already containing the daughter nucleic acids.
Recombination is allowed to occur in the cells and the next
generation of recombinants, or granddaughters are selected by the
methods described above.
[0264] This cycle can be repeated a number of times until the
nucleic acid or peptide having the desired characteristics is
obtained. It is contemplated that in subsequent cycles, the
population of mutated sequences which are added to the preferred
hybrids may come from the parental hybrids or any subsequent
generation.
[0265] In an alternative embodiment, the invention provides a
method of conducting a "molecular" backcross of the obtained
recombinant specific nucleic acid in order to eliminate any neutral
mutations. Neutral mutations are those mutations which do not
confer onto the nucleic acid or peptide the desired properties.
Such mutations may however confer on the nucleic acid or peptide
undesirable characteristics. Accordingly, it is desirable to
eliminate such neutral mutations. The method of this invention
provide a means of doing so.
[0266] In this embodiment, after the hybrid nucleic acid, having
the desired characteristics, is obtained by the methods of the
embodiments, the nucleic acid, the vector having the nucleic acid
or the host cell containing the vector and nucleic acid is
isolated.
[0267] The nucleic acid or vector is then introduced into the host
cell with a large excess of the wild-type nucleic acid. The nucleic
acid of the hybrid and the nucleic acid of the wild-type sequence
are allowed to recombine. The resulting recombinants are placed
under the same selection as the hybrid nucleic acid. Only those
recombinants which retained the desired characteristics will be
selected. Any silent mutations which do not provide the desired
characteristics will be lost through recombination with the
wild-type DNA. This cycle can be repeated a number of times until
all of the silent mutations are eliminated.
[0268] Thus the methods of this invention can be used in a
molecular backcross to eliminate unnecessary or silent
mutations.
[0269] Utility
[0270] The in vivo recombination method of this invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide.
[0271] The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, interleukin I, antibodies, tPA and growth
hormone. This approach may be used to generate proteins having
altered specificity or activity. The approach may also be useful
for the generation of hybrid nucleic acid sequences, for example,
promoter regions, introns, exons, enhancer sequences, 31
untranslated regions or 51 untranslated regions of genes. Thus this
approach may be used to generate genes having increased rates of
expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to
mutate ribozymes or aptamers.
[0272] Scaffold-like regions separating regions of diversity in
proteins may be particularly suitable for the methods of this
invention. The conserved scaffold determines the overall folding by
self-association, while displaying relatively unrestricted loops
that mediate the specific binding. Examples of such scaffolds are
the immunoglobulin beta barrel, and the four-helix bundle. The
methods of this invention can be used to create scaffold-like
proteins with various combinations of mutated sequences for
binding.
[0273] The equivalents of some standard genetic matings may also be
performed by the methods of this invention. For example, a
"molecular" backcross can be performed by repeated mixing of the
hybrid's nucleic acid with the wild-type nucleic acid while
selecting for the mutations of interest. As in traditional
breeding, this approach can be used to combine phenotypes from
different sources into a background of choice. It is useful, for
example, for the removal of neutral mutations that affect
unselected characteristics (ie. immunogenicity). Thus it can be
useful to determine which mutations in a protein are involved in
the enhanced biological activity and which are not.
[0274] Peptide Display Methods The present method can be used to
shuffle, by in vitro and/or in vivo recombination by any of the
disclosed methods, and in any combination, polynucleotide sequences
selected by peptide display methods, wherein an associated
polynucleotide encodes a displayed peptide which is screened for a
phenotype (eg., for affinity for a predetermined receptor
(ligand).
[0275] An increasingly important aspect of bio-pharmaceutical drug
development and molecular biology is the identification of peptide
structures, including the primary amino acid sequences, of peptides
or peptidomimetics that interact with biological macromolecules,
one method of identifying peptides that possess a desired structure
or functional property, such as binding to a predetermined
biological macromolecule (e.g., a receptor), involves the screening
of a large library or peptides for individual library members which
possess the desired structure or functional property conferred by
the amino acid sequence of the peptide.
[0276] In addition to direct chemical synthesis methods for
generating peptide libraries, several recombinant DNA methods also
have been reported. One type involves the display of a peptide
sequence, antibody, or other protein on the surface of a
bacteriophage particle or cell. Generally, in these methods each
bacteriophage particle or cell serves as an individual library
member displaying a single species of displayed peptide in addition
to the natural bacteriophage or cell protein sequences. Each
bacteriophage or cell contains the nucleotide sequence information
encoding the particular displayed peptide sequence; thus, the
displayed peptide sequence can be ascertained by nucleotide
sequence determination of an isolated library member.
[0277] A well-known peptide display method involves the
presentation of a peptide sequence on the surface of a filamentous
bacteriophage, typically as a fusion with a bacteriophage coat
protein. The bacteriophage library can be incubated with an
immobilized, predetermined macromolecule or small molecule (e.g., a
receptor) so that bacteriophage particles which present a peptide
sequence that binds to the immobilized macromolecule can be
differentially partitioned from those that do not present peptide
sequences that bind to the predetermined macromolecule. The
bacteriophage particles (i.e., library members) which are bound to
the immobilized macromolecule are then recovered and replicated to
amplify the selected bacteriophage sub-population for a subsequent
round of affinity enrichment and phage replication. After several
rounds of affinity enrichment and phage replication, the
bacteriophage library members that are thus selected are isolated
and the nucleotide sequence encoding the displayed peptide sequence
is determined, thereby identifying the sequence(s) of peptides that
bind to the predetermined macromolecule (e.g., receptor). Such
methods are further described in PCT patent publication Nos.
91/17271, 91/18980, 91/19818 and 93/08278.
[0278] The latter PCT publication describes a recombinant DNA
method for the display of peptide ligands that involves the
production of a library of fusion proteins with each fusion protein
composed of a first polypeptide portion, typically comprising a
variable sequence, that is available for potential binding to a
predetermined macromolecule, and a second polypeptide portion that
binds to DNA, such as the DNA vector encoding the individual fusion
protein. When transformed host cells are cultured under conditions
that allow for expression of the fusion protein, the fusion protein
binds to the DNA vector encoding it. Upon lysis of the host cell,
the fusion protein/vector DNA complexes can be screened against a
predetermined macromolecule in much the same way as bacteriophage
particles are screened in the phage-based display system, with the
replication and sequencing of the DNA vectors in the selected
fusion protein/vector DNA complexes serving as the basis for
identification of the selected library peptide sequence(s).
[0279] Other systems for generating libraries of peptides and like
polymers have aspects of both the recombinant and in vitro chemical
synthesis methods. In these hybrid methods, cell-free enzymatic
machinery is employed to accomplish the in vitro synthesis of the
library members (ie., peptides or polynucleotides). In one type of
method, RNA molecules with the ability to bind a predetermined
protein or a predetermined dye molecule were selected by alternate
rounds of selection and PCR amplification (Tuerk and Gold, 1990;
Ellington and Szosta{dot over (k)}, 1990). A similar technique was
used to identify DNA sequences which bind a predetermined human
transcription factor (Thiesen and Bach, 1990; Beaudry and Joyce,
1992; PCT patent publication Nos. 92/05258 and 92/14843 *need
inventor). In a similar fashion, the technique of in vitro
translation has been used to synthesize proteins of interest and
has been proposed as a method for generating large libraries of
peptides. These methods which rely upon in vitro translation,
generally comprising stabilized polysome complexes, are described
further in PCT patent publication Nos. 88/08453, 90/05785,
90/07003, 91/02076, 91/05058, and 92/02536. Applicants have
described methods in which library members comprise a fusion
protein having a first polypeptide portion with DNA binding
activity and a second polypeptide portion having the library member
unique peptide sequence; such methods are suitable for use in
cell-free in vitro selection formats, among others.
[0280] The displayed peptide sequences can be of varying lengths,
typically from 3-5000 amino acids long or longer, frequently from
5-100 amino acids long, and often from about 8-15 amino acids long.
A library can comprise library members having varying lengths of
displayed peptide sequence, or may comprise library members having
a fixed length of displayed peptide sequence. Portions or all of
the displayed peptide sequence(s) can be random, pseudorandom,
defined set kernal, fixed, or the like. The present display methods
include methods for in vitro and in vivo display of single-chain
antibodies, such as nascent scFv on polysomes or scfv displayed on
phage, which enable large-scale screening of scfv libraries having
broad diversity of variable region sequences and binding
specificities.
[0281] The present invention also provides random, pseudorandom,
and defined sequence framework peptide libraries and methods for
generating and screening those libraries to identify useful
compounds (e.g., peptides, including single-chain antibodies) that
bind to receptor molecules or epitopes of interest or gene products
that modify peptides or RNA in a desired fashion. The random,
pseudorandom, and defined sequence framework peptides are produced
from libraries of peptide library members that comprise displayed
peptides or displayed single-chain antibodies attached to a
polynucleotide template from which the displayed peptide was
synthesized. The mode of attachment may vary according to the
specific embodiment of the invention selected, and can include
encapsulation in a phage particle or incorporation in a cell.
[0282] A method of affinity enrichment allows a very large library
of peptides and single-chain antibodies to be screened and the
polynucleotide sequence encoding the desired peptide(s) or
single-chain antibodies to be selected. The polynucleotide can then
be isolated and shuffled to recombine combinatorially the amino
acid sequence of the selected peptide(s) (or predetermined portions
thereof) or single-chain antibodies (or just VHI, VLI or CDR
portions thereof). Using these methods, one can identify a peptide
or single-chain antibody as having a desired binding affinity for a
molecule and can exploit the process of shuffling to converge
rapidly to a desired high-affinity peptide or scfv. The peptide or
antibody can then be synthesized in bulk by conventional means for
any suitable use (e.g., as a therapeutic or diagnostic agent).
[0283] A significant advantage of the present invention is that no
prior information regarding an expected ligand structure is
required to isolate peptide ligands or antibodies of interest. The
peptide identified can have biological activity, which is meant to
include at least specific binding affinity for a selected receptor
molecule and, in some instances, will further include the ability
to block the binding of other compounds, to stimulate or inhibit
metabolic pathways, to act as a signal or messenger, to stimulate
or inhibit cellular activity, and the like.
[0284] The present invention also provides a method for shuffling a
pool of polynucleotide sequences selected by affinity screening a
library of polysomes displaying nascent peptides (including
single-chain antibodies) for library members which bind to a
predetermined receptor (e.g., a mammalian proteinaceous receptor
such as, for example, a peptidergic hormone receptor, a cell
surface receptor, an intracellular protein which binds to other
protein(s) to form intracellular protein complexes such as
hetero-dimers and the like) or epitope (e.g., an immobilized
protein, glycoprotein, oligosaccharide, and the like).
[0285] Polynucleotide sequences selected in a first selection round
(typically by affinity selection for binding to a receptor (e.g., a
ligand)).by any of these methods are pooled and the pool(s) is/are
shuffled by in vitro and/or in vivo recombination to produce a
shuffled pool comprising a population of recombined selected
polynucleotide sequences. The recombined selected polynucleotide
sequences are subjected to at least one subsequent selection round.
The polynucleotide sequences selected in the subsequent selection
round(s) can be used directly, sequenced, and/or subjected to one
or more additional rounds of shuffling and subsequent selection.
Selected sequences can also be back-crossed with polynucleotide
sequences encoding neutral sequences (i.e., having insubstantial
functional effect on binding), such as for example by back-crossing
with a wild-type or naturally-occurring sequence substantially
identical to a selected sequence to produce native-like functional
peptides, which may be less immunogenic. Generally, during
back-crossing subsequent selection is applied to retain the
property of binding to the predetermined receptor (ligand).
[0286] Prior to or concomitant with the shuffling of selected
sequences, the sequences can be mutagenized. In one embodiment,
selected library members are cloned in a prokaryotic vector (e.g.,
plasmid, phagemid, or bacteriophage) wherein a collection of
individual. colonies (or plaques) representing discrete library
members are produced. Individual selected library members can then
be manipulated (e.g., by site-directed mutagenesis, cassette
mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like)
to generate a collection of library members representing a kernal
of sequence diversity based on the sequence of the selected library
member. The sequence of an individual selected library member or
pool can be manipulated to incorporate random mutation,
pseudorandom mutation, defined kernal mutation (i e., comprising
variant and invariant residue positions and/or comprising variant
residue positions which can comprise a residue selected from a
defined subset of amino acid residues), codon-based mutation, and
the like, either segmentally or over the entire length of the
individual selected library member sequence. The mutagenized
selected library members are then shuffled by in vitro and/or in
vivo recombinatorial shuffling as disclosed herein.
[0287] The invention also provides peptide libraries comprising a
plurality of individual library members of the invention, wherein
(1) each individual library member of said plurality comprises a
sequence produced by shuffling of a pool of selected sequences, and
(2) each individual library member comprises a variable peptide
segment sequence or single-chain antibody segment sequence which is
distinct from the variable peptide segment sequences or
single-chain antibody sequences of other individual library members
in said plurality (although some library members may be present in
more than one copy per library due to uneven amplification,
stochastic probability or the like).
[0288] The invention also provides a product-by-process, wherein
selected polynucleotide sequences having (or encoding a peptide
having) a predetermined binding specificity are formed by the
process of: (1) screening a displayed peptide or displayed
single-chain antibody library against a predetermined receptor
(e.g., ligand) or epitope (e.g., antigen macromolecule) and
identifying and/or enriching library members which bind to the
predetermined receptor or epitope to produce a pool of selected
library members, (2) shuffling by recombination the selected
library members (or amplified or cloned copies thereof) which binds
the predetermined epitope and has been thereby isolated and/or
enriched from the library to generate a shuffled library, and (3)
screening the shuffled library against the predetermined receptor
(e.g., ligand) or epitope (e.g., antigen macromolecule) and
identifying and/or enriching shuffled library members which bind to
the predetermined receptor or epitope to produce a pool of selected
shuffled library members.
[0289] Antibody Display and Screening Methods
[0290] The present method can be used to shuffle, by in vitro
and/or in vivo recombination by any of the disclosed methods, and
in any combination, polynucleotide sequences selected by antibody
display methods, wherein an associated polynucleotide encodes a
displayed antibody which is screened for a phenotype (e.g., for
affinity for binding a predetermined antigen (ligand).
[0291] Various molecular genetic approaches have been devised to
capture the vast immunological repertoire represented by the
extremely large number of distinct variable regions which can be
present in immunoglobulin chains. The naturally-occurring germ line
immunoglobulin heavy chain locus is composed of separate tandem
arrays of variable segment genes located upstream of a tandem array
of diversity segment genes, which are themselves located upstream
of a tandem array of joining (i) region genes, which are located
upstream of the constant region genes. During B lymphocyte
development, V-D-J rearrangement occurs wherein a heavy chain
variable region gene (VH) is formed by rearrangement to form a
fused D segment followed by rearrangement with a V segment to form
a V-D-J joined product gene which, if productively rearranged,
encodes a functional variable region (VH) of a heavy chain.
Similarly, light chain loci rearrange one of several V segments
with one of several J segments to form a gene encoding the variable
region (VL) of a light chain.
[0292] The vast repertoire of variable regions possible in
immunoglobulins derives in part from the numerous combinatorial
possibilities of joining V and i segments (and, in the case of
heavy chain loci, D segments) during rearrangement in B cell
development. Additional sequence diversity in the heavy chain
variable regions arises from non-uniform rearrangements of the D
segments during V-D-J joining and from N region addition. Further,
antigen-selection of specific B cell clones selects for higher
affinity variants having non-germline mutations in one or both of
the heavy and light chain variable regions; a phenomenon referred
to as "affinity maturation" or "affinity sharpening". Typically,
these "affinity sharpening" mutations cluster in specific areas of
the variable region, most commonly in the
complementarity-determining regions (CDRs).
[0293] In order to overcome many of the limitations in producing
and identifying high-affinity immunoglobulins through
antigen-stimulated .beta. cell development (i.e., immunization),
various prokaryotic expression systems have been developed that can
be manipulated to produce combinatorial antibody libraries which
may be screened for high-affinity antibodies to specific antigens.
Recent advances in the expression of antibodies in Escherichia coli
and bacteriophage systems (see, *"Alternative Peptide Display
Methods", infra) have raised the possibility that virtually any
specificity can be obtained by either cloning antibody genes from
characterized hybridomas or by de novo selection using antibody
gene libraries (e.g., from Ig CDNA).
[0294] Combinatorial libraries of antibodies have been generated in
bacteriophage lambda expression systems which may be screened as
bacteriophage plaques or as colonies of lysogens (Huse et al,.
1989); Caton and Koprowski, 1990; Mullinax et al., 1990; Persson et
al., 1991). Various embodiments of bacteriophage antibody display
libraries and lambda phage expression libraries have been described
(Kang et al., 1991; Clackson et al., 1991; McCafferty et al., 1990;
Burton et al., 1991; Hoogenboom et al., 1991; Chang et al., 1991;
Breitling et al., 1991; Marks et al., 1991, p.581; Barbas et al.,
1992; Hawkins and Winter, 1992; Marks et al., 1992, p. 779; Marks
et al., 1992, p. 16007; Lowman et al., 1991; Lerner et al., 1992,
incorporated herein by reference). Typically, a bacteriophage
antibody display library is screened with a receptor (e.g.,
polypeptide, carbohydrate, glycoprotein, nucleic acid) that is
immobilized (e.g., by covalent linkage to a chromatography resin to
enrich for reactive phage by affinity chromatography) and/or
labeled (e.g., to screen plaque or colony lifts).
[0295] One particularly advantageous approach has been the use of
so-called single-chain fragment variable (scfv) libraries (Marks et
al., supra, p. 779; Winter and Milstein, 1991; Clackson et al.,
supra; Marks et al., supra, p. 581; Chaudhary et al., 1990;
Chiswell et al., 1992; McCafferty et al., supra; and Huston et al.,
1988). Various embodiments of scfv libraries displayed on
bacteriophage coat proteins have been described.
[0296] Beginning in 1988, single-chain analogues of Fv fragments
and their fusion proteins have been reliably generated by antibody
engineering methods. The first step generally involves obtaining
the genes encoding VH and VL domains with desired binding
properties; these V genes may be isolated from a specific hybridoma
cell line, selected from a combinatorial V-gene library, or made by
V gene synthesis. The single-chain Fv is formed by connecting the
component V genes with an oligonucleotide that encodes an
appropriately designed linker peptide, such as
(Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s). The linker
bridges the C-terminus of the first V region and N-terminus of the
second, ordered as either VH-linker-VL or VL-linker-VH' In
principle, the scfv binding site can faithfully replicate both the
affinity and specificity of its parent antibody combining site.
[0297] Thus, scfv fragments are comprised of VH and VL domains
linked into a single polypeptide chain by a flexible linker
peptide. After the scfv genes are assembled, they are cloned into a
phagemid and expressed at the tip of the M13 phage (or similar
filamentous bacteriophage) as fusion proteins with the
bacteriophage PIII (gene 3) coat protein. Enriching for phage
expressing an antibody of interest is accomplished by panning the
recombinant phage displaying a population scfv for binding to a
predetermined epitope (e.g., target antigen, receptor).
[0298] The linked polynucleotide of a library member provides the
basis for replication of the library member after a screening or
selection procedure, and also provides the basis for the
determination, by nucleotide sequencing, of the identity of the
displayed peptide sequence or VH and VL amino acid sequence. The
displayed peptide (s) or single-chain antibody (e. g., scfv) and/or
its VH and VL domains or their CDRs can be cloned and expressed in
a suitable expression system. Often polynucleotides encoding the
isolated VH and VL domains will be ligated to polynucleotides
encoding constant regions (CH and CL) to form polynucleotides
encoding complete antibodies (e.g., chimeric or fully-human),
antibody fragments, and the like. Often polynucleotides encoding
the isolated CDRs will be grafted into polynucleotides encoding a
suitable variable region framework (and optionally constant
regions) to form polynucleotides encoding complete antibodies
(e.g., humanized or fully-human), antibody fragments, and the like.
Antibodies can be used to isolate preparative quantities of the
antigen by immunoaffinity chromatography. Various other uses of
such antibodies are to diagnose and/or stage disease (e.g.,
neoplasia) and for therapeutic application to treat disease, such
as for example: neoplasia, autoimmune disease, AIDS, cardiovascular
disease, infections, and the like.
[0299] Various methods have been reported for increasing the
combinatorial diversity of a scfv library to broaden the repertoire
of binding species (idiotype spectrum) The use of PCR has permitted
the variable regions to be rapidly cloned either from a specific
hybridoma source or as a gene library from non-immunized cells,
affording combinatorial diversity in the assortment of VH and VL
cassettes which can be combined. Furthermore, the VH and VL
cassettes can themselves be diversified, such as by random,
pseudorandom, or directed mutagenesis. Typically, VH and VL
cassettes are diversified in or near the
complementarity-determining regions (CDRS), often the third CDR,
CDR3. Enzymatic inverse PCR mutagenesis has been shown to be a
simple and reliable method for constructing relatively large
libraries of scfv site-directed hybrids (Stemmer el al., 1993), as
has error-prone PCR and chemical mutagenesis (Deng et al., 1994).
Riechmann et al. (1993) Biochemistry 32:8848 showed semi-rational
design of an antibody scfv fragment using site-directed
randomization by degenerate oligonucleotide PCR and subsequent
phage display of the resultant scfv hybrids. * Barbas et al. (1992)
on.cit. attempted to circumvent the problem of limited repertoire
sizes resulting from using biased variable region sequences by
randomizing the sequence in a synthetic CDR region of a human
tetanus toxoid-binding Fab.
[0300] CDR randomization has the potential to create approximately
1.times.10.sup.20 CDRs for the heavy chain CDR3 alone, and a
roughly similar number of variants of the heavy chain CDR1 and
CDR2, and light chain CDR1-3 variants. Taken individually or
together, the combination possibilities of CDR randomization of
heavy and/or light chains requires generating a prohibitive number
of bacteriophage clones to produce a clone library representing all
possible combinations, the vast majority of which will be
non-binding. Generation of such large numbers of primary
transformants is not feasible with current transformation
technology and bacteriophage display systems. For example, Barbas
et al. (1992) op.cit. only generated 5.times.10.sup.7
transformants, which represents only a tiny fraction of the
potential diversity of a library of thoroughly randomized CDRS.
[0301] Despite these substantial limitations, bacteriophage display
of scfv have already yielded a variety of useful antibodies and
antibody fusion proteins. A bispecific single chain antibody has
been shown to mediate efficient tumor cell lysis (Gruber et al.,
1994). Intracellular expression of an anti-Rev scfv has been shown
to inhibit HIV-1 virus replication in vitro (Duan et al., 1994),
and intracellular expression of an anti-p2lrar, scfv has been shown
to inhibit meiotic maturation of Xenopus oocytes (Biocca et al.,
1993). Recombinant scfv which can be used to diagnose HIV infection
have also been reported, demonstrating the diagnostic utility of
scfv (Lilley et al., 1994). Fusion proteins wherein an scFv is
linked to a second polypeptide, such as a toxin or fibrinolytic
activator protein, have also been reported (Holvost et al., 1992;
Nicholls et al., 1993).
[0302] If it were possible to generate scfv libraries having
broader antibody diversity and overcoming many of the limitations
of conventional CDR mutagenesis and randomization methods which can
cover only a very tiny fraction of the potential sequence
combinations, the number and quality of scfv antibodies suitable
for therapeutic and diagnostic use could be vastly improved. To
address this, the in vitro and in vivo shuffling methods of the
invention are used to recombine CDRs which have been obtained
(typically via PCR amplification or cloning) from nucleic acids
obtained from selected displayed antibodies. Such displayed
antibodies can be displayed on cells, on bacteriophage particles,
on polysomes, or any suitable antibody display system wherein the
antibody is associated with its encoding nucleic acid(s). In a
variation, the CDRs are initially obtained from MRNA (or CDNA) from
antibody-producing cells (e.g., plasma cells/splenocytes from an
immunized wild-type mouse, a human, or a transgenic mouse capable
of making a human antibody as in W092/03918, W093/12227, and
W094/25585), including hybridomas derived therefrom.
[0303] Polynucleotide sequences selected in a first selection round
(typically by affinity selection for displayed antibody binding to
an antigen (e.g., a ligand) by any of these methods are pooled and
the pool(s) is/are shuffled by in vitro and/or in vivo
recombination, especially shuffling of CDRs (typically shuffling
heavy chain CDRs with other heavy chain CDRs and light chain CDRs
with other light chain CDRS) to produce a shuffled pool comprising
a population of recombined selected polynucleotide sequences. The
recombined selected polynucleotide sequences are expressed in a
selection format as a displayed antibody and subjected to at least
one subsequent selection round. The polynucleotide sequences
selected in the subsequent selection round(s) can be used directly,
sequenced, and/or subjected to one or more additional rounds of
shuffling and subsequent selection until an antibody of the desired
binding affinity is obtained. Selected sequences can also be
back-crossed with polynucleotide sequences encoding neutral
antibody framework sequences (i.e., having insubstantial functional
effect on antigen binding), such as for example by back-crossing
with a human variable region framework to produce human-like
sequence antibodies. Generally, during back-crossing subsequent
selection is applied to retain the property of binding to the
predetermined antigen.
[0304] Alternatively, or in combination with the noted variations,
the valency of the target epitope may be varied to control the
average binding affinity of selected scfv library members. The
target epitope can be bound to a surface or substrate at varying
densities, such as by including a competitor epitope, by dilution,
or by other method known to those in the art. A high density
(valency) of predetermined epitope can be used to enrich for scfv
library members which have relatively low affinity, whereas a low
density (valency) can preferentially enrich for higher affinity
scfv library members.
[0305] For generating diverse variable segments, a collection of
synthetic oligonucleotides encoding random, pseudorandom, or a
defined sequence kernal set of peptide sequences can be inserted by
ligation into a predetermined site (e.g., a CDR). Similarly, the
sequence diversity of one or more CDRs of the single-chain antibody
cassette(s) can be expanded by mutating the CDR(s) with
site-directed mutagenesis, CDR-replacement, and the like. The
resultant DNA molecules can be propagated in a host for cloning and
amplification prior to shuffling, or can be used directly (i.e.,
may avoid loss of diversity which may occur upon propagation in a
host cell) and the selected library members subsequently
shuffled.
[0306] Displayed peptide/polynucleotide complexes (library members)
which encode a variable segment peptide sequence of interest or a
single-chain antibody of interest are selected from the library by
an affinity enrichment technique. This is accomplished by means of
a immobilized macromolecule or epitope specific for the peptide
sequence of interest, such as a receptor, other macromolecule, or
other epitope species. Repeating the affinity selection procedure
provides an enrichment of library members encoding the desired
sequences, which may then be isolated for pooling and shuffling,
for sequencing, and/or for further propagation and affinity
enrichment.
[0307] The library members without the desired specificity are
removed by washing. The degree and stringency of washing required
will be determined for each peptide sequence or single-chain
antibody of interest and the immobilized predetermined
macromolecule or epitope. A certain degree of control can be
exerted over the binding characteristics of the nascent peptide/DNA
complexes recovered by adjusting the conditions of the binding
incubation and the subsequent washing. The temperature, pH, ionic
strength, divalent cations concentration, and the volume and
duration of the washing will select for nascent peptide/DNA
complexes within particular ranges of affinity for the immobilized
macromolecule. Selection based on slow dissociation rate, which is
usually predictive of high affinity, is often the most practical
route. This may be done either by continued incubation in the
presence of a saturating amount of free predetermined
macromolecule, or by increasing the volume, number, and length of
the washes. In each case, the rebinding of dissociated nascent
peptide/DNA or peptide/RNA complex is prevented, and with
increasing time, nascent peptide/DNA or peptide/RNA complexes of
higher and higher affinity are recovered.
[0308] Additional modifications of the binding and washing
procedures may be applied to find peptides with special
characteristics. The affinities of some peptides are dependent on
ionic strength or cation concentration. This is a useful
characteristic for peptides that will be used in affinity
purification of various proteins when gentle conditions for
removing the protein from the peptides are required.
[0309] One variation involves the use of multiple binding targets
(multiple epitope species, multiple receptor species), such that a
scfv library can be simultaneously screened for a multiplicity of
scfv which have different binding specificities. Given that the
size of a scfv library often limits the diversity of potential scfv
sequences, it is typically desirable to us scfv libraries of as
large a size as possible. The time and economic considerations of
generating a number of very large polysome scFv-display libraries
can become prohibitive. To avoid this substantial problem, multiple
predetermined epitope species (receptor species) can be
concomitantly screened in a single library, or sequential screening
against a number of epitope species can be used. In one variation,
multiple target epitope species, each encoded on a separate bead
(or subset of beads), can be mixed and incubated with a
polysome-display scfv library under suitable binding conditions.
The collection of beads, comprising multiple epitope species, can
then be used to isolate, by affinity selection, scfv library
members. Generally, subsequent affinity screening rounds can
include the same mixture of beads, subsets thereof, or beads
containing only one or two individual epitope species. This
approach affords efficient screening, and is compatible with
laboratory automation, batch processing, and high throughput
screening methods.
[0310] A variety of techniques can be used in the present invention
to diversify a peptide library or single-chain antibody library, or
to diversify, prior to or concomitant with shuffling, around
variable segment peptides found in early rounds of panning to have
sufficient binding activity to the predetermined macromolecule or
epitope. In one approach, the positive selected
peptide/polynucleotide complexes (those identified in an early
round of affinity enrichment) are sequenced to determine the
identity of the active peptides. Oligonucleotides are then
synthesized based on these active peptide sequences, employing a
low level of all bases incorporated at each step to produce slight
variations of the primary oligonucleotide sequences. This mixture
of (slightly) degenerate oligonucleotides is then cloned into the
variable segment sequences at the appropriate locations. This
method produces systematic, controlled variations of the starting
peptide sequences, which can then be shuffled. It requires,
however, that individual positive nascent peptide/polynucleotide
complexes be sequenced before mutagenesis, and thus is useful for
expanding the diversity of small numbers of recovered complexes and
selecting variants having higher binding affinity and/or higher
binding specificity. In a variation, mutagenic PCR amplification of
positive selected peptide/polynucleotide complexes (especially of
the variable region sequences, the amplification products of which
are shuffled in vitro and/or in vivo and one or more additional
rounds of screening is done prior to sequencing. The same general
approach can be employed with single-chain antibodies in order to
expand the diversity and enhance the binding affinity/specificity,
typically by diversifying CDRs or adjacent framework regions prior
to or concomitant with shuffling. If desired, shuffling reactions
can be spiked with mutagenic oligonucleotides capable of in vitro
recombination with the selected library members can be included.
Thus, mixtures of synthetic oligonucleotides and PCR produced
polynucleotides (synthesized by error-prone or high-fidelity
methods) can be added to the in vitro shuffling mix and be
incorporated into resulting shuffled library members
(shufflants).
[0311] The present invention of shuffling enables the generation of
a vast library of CDR-variant single-chain antibodies. One way to
generate such antibodies is to insert synthetic CDRs into the
single-chain antibody and/or CDR randomization prior to or
concomitant with shuffling. The sequences of the synthetic CDR
cassettes are selected by referring to known sequence data of human
CDR and are selected in the discretion of the practitioner
according to the following guidelines: synthetic CDRs will have at
least 40 percent positional sequence identity to known CDR
sequences, and preferably will have at least 50 to 70 percent
positional sequence identity to known CDR sequences. For example, a
collection of synthetic CDR sequences can be generated by
synthesizing a collection of oligonucleotide sequences on the basis
of naturally-occurring human CDR sequences listed in Kabat et al.
(1991) op.cit.; the pool (s) of synthetic CDR sequences are
calculated to encode CDR peptide sequences having at least 40
percent sequence identity to at least one known naturally-occurring
human CDR sequence. Alternatively, a collection of
naturally-occurring CDR sequences may be compared to generate
consensus sequences so that amino acids used at a residue position
frequently (i.e., in at least 5 percent of known CDR sequences) are
incorporated into the synthetic CDRs at the corresponding
position(s). Typically, several (e.g., 3 to about 50) known CDR
sequences are compared and observed natural sequence variations
between the known CDRs are tabulated, and a collection of
oligonucleotides encoding CDR peptide sequences encompassing all or
most permutations of the observed natural sequence variations is
synthesized. For example but not for limitation, if a collection of
human VH CDR sequences have carboxy-terminal amino acids which are
either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR
oligonucleotide sequences are designed to allow the
carboxy-terminal CDR residue to be any of these amino acids. In
some embodiments, residues other than those which naturally-occur
at a residue position in the collection of CDR sequences are
incorporated: conservative amino acid substitutions are frequently
incorporated and up to 5 residue positions may be varied to
incorporate non-conservative amino acid substitutions as compared
to known naturally-occurring CDR sequences. Such CDR sequences can
be used in primary library members (prior to first round screening)
and/or can be used to spike in vitro shuffling reactions of
selected library member sequences. Construction of such pools of
defined and/or degenerate sequences will be readily accomplished by
those of ordinary skill in the art.
[0312] The collection of synthetic CDR sequences comprises at least
one member that is not known to be a naturally-occurring CDR
sequence. It is within the discretion of the practitioner to
include or not include a portion of random or pseudorandom sequence
corresponding to N region addition in the heavy chain CDR; the N
region sequence ranges from 1 nucleotide to about 4 nucleotides
occurring at V-D and D-J junctions. A collection of synthetic heavy
chain CDR sequences comprises at least about 100 unique CDR
sequences, typically at least about 1,000 unique CDR sequences,
preferably at least about 10,000 unique CDR sequences, frequently
more than 50,000 unique CDR sequences; however, usually not more
than about 1.times.10 6 unique CDR sequences are included in the
collection, although occasionally 1.times.107 to 1.times.108 unique
CDR sequences are present, especially if conservative amino acid
substitutions are permitted at positions where the conservative
amino acid substituent is not present or is rare (i.e., less than
0.1 percent) in that position in naturally-occurring human CDRS. In
general, the number of unique CDR sequences included in a library
should not exceed the expected number of primary transformants in
the library by more than a factor of 10. Such single-chain
antibodies generally bind of about at least 1.times.10 m-,
preferably with an affinity of about at least 5.times.10.sup.7 M-1,
more preferably with an affinity of at least 1.times.10.sup.8 M-1
to 1.times.10.sup.9 M-1 or more, sometimes up to 1.times.10.sup.10
M-1 or more. Frequently, the predetermined antigen is a human
protein, such as for example a human cell surface antigen (e. g.,
CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human
biological macromolecule (e.g., thrombomodulin, protein C,
carbohydrate antigen, sialyl Lewis antigen, Lselectin), or nonhuman
disease associated macromolecule (e.g., bacterial LPS, virion
capsid protein or envelope glycoprotein) and the like.
[0313] High affinity single-chain antibodies of the desired
specificity can be engineered and expressed in a variety of
systems. For example, scfv have been produced in plants (Firek et
al., 1993) and can be readily made in prokaryotic systems (Owens
and Young, 1994; Johnson and Bird, 1991). Furthermore, the
single-chain antibodies can be used as a basis for constructing
whole antibodies or various fragments thereof (Kettleborough et
al., 1994). The variable region encoding sequence may be isolated
(e.g., by PCR amplification or subcloning) and spliced to a
sequence encoding a desired human constant region to encode a human
sequence antibody more suitable for human therapeutic uses where
immunogenicity is preferably minimized. The polynucleotide(s)
having the resultant fully human encoding sequence(s) can be
expressed in a host cell (e.g., from an expression vector in a
mammalian cell) and purified for pharmaceutical formulation.
[0314] The DNA expression constructs will typically include an
expression control DNA sequence operably linked to the coding
sequences, including naturally-associated or heterologous promoter
regions. Preferably, the expression control sequences will be
eukaryotic promoter systems in vectors capable of transforming or
transfecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and the collection and purification of the
mutant` "engineered" antibodies.
[0315] As stated previously, the DNA sequences will be expressed in
hosts after the sequences have been operably linked to an
expression control sequence (i.e., positioned to ensure the
transcription and translation of the structural gene). These
expression vectors are typically replicable in the host organisms
either as episomes or as an integral part of the host chromosomal
DNA. Commonly, expression vectors will contain selection markers,
e.g., tetracycline or neomycin, to permit detection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362, which is incorporated herein by reference).
[0316] In addition to eukaryotic microorganisms such as yeast,
mammalian tissue cell culture may also be used to produce the
polypeptides of the present invention (see, Winnacker, 1987), which
is incorporated herein by reference). Eukaryotic cells are actually
preferred, because a number of suitable host cell lines capable of
secreting intact immunoglobulins have been developed in the art,
and include the CHO cell lines, various COS cell lines, HeLa cells,
and myeloma cell lines, but preferably transformed Bcells or
hybridomas. Expression vectors for these cells can include
expression control sequences, such as an origin of replication, a
promoter, an enhancer (Queen et al., 1986), and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites, and transcriptional terminator
sequences. Preferred expression control sequences are promoters
derived from immunoglobulin genes, cytomegalovirus, SV40,
Adenovirus, Bovine Papilloma Virus, and the like.
[0317] Eukaryotic DNA transcription can be increased by inserting
an enhancer sequence into the vector. Enhancers are cis-acting
sequences of between 10 to 300 bp that increase transcription by a
promoter. Enhancers can effectively increase transcription when
either 51 or 31 to the transcription unit. They are also effective
if located within an intron or within the coding sequence itself.
Typically, viral enhancers are used, including SV40 enhancers,
cytomegalovirus enhancers, polyoma enhancers, and adenovirus
enhancers. Enhancer sequences from mammalian systems are also
commonly used, such as the mouse immunoglobulin heavy chain
enhancer.
[0318] Mammalian expression vector systems will also typically
include a selectable marker gene. Examples of suitable markers
include, the dihydrofolate reductase gene (DHFR), the thymidine
kinase gene (TK), or prokaryotic genes conferring drug resistance.
The first two marker genes prefer the use of mutant cell lines that
lack the ability to grow without the addition of thymidine to the
growth medium. Transformed cells can then be identified by their
ability to grow on non-supplemented media. Examples of prokaryotic
drug resistance genes useful as markers include genes conferring
resistance to G418, mycophenolic acid and hygromycin.
[0319] The vectors containing the DNA segments of interest can be
transferred into the host cell by well-known methods, depending on
the type of cellular host. For example, calcium chloride
transfection is commonly utilized for prokaryotic cells, whereas
calcium phosphate treatment. lipofection, or electroporation may be
used for other cellular hosts. Other methods used to transform
mammalian cells include the use of Polybrene, protoplast fusion,
liposomes, electroporation, and micro-injection (see, generally,
Sambrook et al., supra).
[0320] Once expressed, the antibodies, individual mutated
immunoglobulin chains, mutated antibody fragments, and other
immunoglobulin polypeptides of the invention can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, fraction column chromatography, gel
electrophoresis and the like (see, generally, Scopes, 1982). Once
purified, partially or to homogeneity as desired, the polypeptides
may then be used therapeutically or in developing and performing
assay procedures, immunofluorescent stainings, and the like (see,
generally, Lefkovits and Pernis, 1979 and 1981).
[0321] The antibodies generated by the method of the present
invention can be used for diagnosis and therapy. By way of
illustration and not limitation, they can be used to treat cancer,
autoimmune diseases, or viral infections. For treatment of cancer,
the antibodies will typically bind to an antigen expressed
preferentially on cancer cells, such as erbB-2, CEA, CD33, and many
other antigens and binding members well known to those skilled in
the art.
[0322] Two-Hybrid Based Screening Assays
[0323] Shuffling can also be used to recombinatorially diversify a
pool of selected library members obtained by screening a two-hybrid
screening system to identify library members which bind a
predetermined polypeptide sequence. The selected library members
are pooled and shuffled by in vitro and/or in vivo recombination.
The shuffled pool can then be screened in a yeast two hybrid system
to select library members which bind said predetermined polypeptide
sequence (e. g., and SH2 domain) or which bind an alternate
predetermined polypeptide sequence (e.g., an SH2 domain from
another protein species).
[0324] An approach to identifying polypeptide sequences which bind
to a predetermined polypeptide sequence has been to use a so-called
"two-hybrid" system wherein the predetermined polypeptide sequence
is present in a fusion protein (Chien et al., 1991). This approach
identifies protein-protein interactions in vivo through
reconstitution of a transcriptional activator (Fields and Song,
1989), the yeast Gal4 transcription protein. Typically, the method
is based on the properties of the yeast Gal4 protein, which
consists of separable domains responsible for DNA-binding and
transcriptional activation. Polynucleotides encoding two hybrid
proteins, one consisting of the yeast Gal4 DNA-binding domain fused
to a polypeptide sequence of a known protein and the other
consisting of the Gal4 activation domain fused to a polypeptide
sequence of a second protein, are constructed and introduced into a
yeast host cell. Intermolecular binding between the two fusion
proteins reconstitutes the Gal4 DNA-binding domain with the Gal4
activation domain, which leads to the transcriptional activation of
a reporter gene (e.g., lacz, HIS3) which is operably linked to a
Gal4 binding site. Typically, the two-hybrid method is used to
identify novel polypeptide sequences which interact with a known
protein (Silver and Hunt, 1993; Durfee et al., 1993; Yang et al.,
1992; Luban et al., 1993; Hardy et al., 1992; Bartel et al., 1993;
and Vojtek et al., 1993). However, variations of the two-hybrid
method have been used to identify mutations of a known protein that
affect its binding to a second known protein (Li and Fields, 1993;
Lalo et al., 1993; Jackson et al., 1993; and Madura et al., 1993).
Two-hybrid systems have also been used to identify interacting
structural domains of two known proteins (Bardwell et al., 1993;
Chakrabarty et al., 1992; Staudinger et al., 1993; and Milne and
Weaver 1993) or domains responsible for oligomerization of a single
protein (Iwabuchi et al., 1993; Bogerd et al., 1993). Variations of
two-hybrid systems have been used to study the in vivo activity of
a proteolytic enzyme (Dasmahapatra et al., 1992). Alternatively, an
E. coli/BCCP interactive screening system (Germino et al., 1993;
Guarente, 1993) can be used to identify interacting protein
sequences (i.e., protein sequences which heterodimerize or form
higher order heteromultimers). Sequences selected by a two-hybrid
system can be pooled and shuffled and introduced into a two-hybrid
system for one or more subsequent rounds of screening to identify
polypeptide sequences which bind to the hybrid containing the
predetermined binding sequence. The sequences thus identified can
be compared to identify consensus sequence(s) and consensus
sequence kernals.
[0325] In general, standard techniques of recombination DNA
technology are described in various publications. e.g. Sambrook et
al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory; Ausubel et al., 1987, Current Protocols in
Molecular Biology, vols. 1 and 2 and supplements, and Berger and
Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular
Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.,
each of which is incorporated herein in their entirety by
reference. Polynucleotide modifying enzymes were used according to
the manufacturer's recommendations. Oligonucleotides were
synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizer
using ABI chemicals. If desired, PCR amplimers for amplifying a
predetermined DNA sequence may be selected at the discretion of the
practitioner.
[0326] One microgram samples of template DNA are obtained and
treated with U.V. light to cause the formation of dimers, including
TT dimers, particularly purine dimers. U.V. exposure is limited so
that only a few photoproducts are generated per gene on the
template DNA sample. Multiple samples are treated with U.V. light
for varying periods of time to obtain template DNA samples with
varying numbers of dimers from U.V. exposure.
[0327] A random priming kit which utilizes a non-proofreading
polymease (for example, Prime-It II Random Primer Labeling kit by
Stratagene Cloning Systems) is utilized to generate different size
polynucleotides by priming at random sites on templates which are
prepared by U.V. light (as described above) and extending along the
templates. The priming protocols such as described in the Prime-It
II Random Primer Labeling kit may be utilized to extend the
primers. The dimers formed by U.V. exposure serve as a roadblock
for the extension by the non-proofreading polymerase. Thus, a pool
of random size polynucleotides is present after extension with the
random primers is finished.
[0328] The present invention is further directed to a method for
generating a selected mutant polynucleotide sequence (or a
population of selected polynucleotide sequences) typically in the
form of amplified and/or cloned polynucleotides, whereby the
selected polynucleotide sequences(s) possess at least one desired
phenotypic characteristic (e.g., encodes a polypeptide, promotes
transcription of linked polynucleotides, binds a protein, and the
like) which can be selected for. One method for identifying hybrid
polypeptides that possess a desired structure or functional
property, such as binding to a predetermined biological
macromolecule (e.g., a receptor), involves the screening of a large
library of polypeptides for individual library members which
possess the desired structure or functional property conferred by
the amino acid sequence of the polypeptide.
[0329] In one embodiment, the present invention provides a method
for generating libraries of displayed polypeptides or displayed
antibodies suitable for affinity interaction screening or
phenotypic screening. The method comprises (1) obtaining a first
plurality of selected library members comprising a displayed
polypeptide or displayed antibody and an associated polynucleotide
encoding said displayed polypeptide or displayed antibody, and
obtaining said associated polynucleotides or copies thereof wherein
said associated polynucleotides comprise a region of substantially
identical sequences, optimally introducing mutations into said
polynucleotides or copies, (2) pooling the polynucleotides or
copies, (3) producing smaller or shorter polynucleotides by
interrupting a random or particularized priming and synthesis
process or an amplification process, and (4) performing
amplification, preferably PCR amplification, and optionally
mutagenesis to homologously recombine the newly synthesized
polynucleotides.
[0330] It is a particularly preferred object of the invention to
provide a process for producing hybrid polynucleotides which
express a useful hybrid polypeptide by a series of steps
comprising:
[0331] (a) producing polynucleotides by interrupting a
polynucleotide amplification or synthesis process with a means for
blocking or interrupting the amplification or synthesis process and
thus providing a plurality of smaller or shorter polynucleotides
due to the replication of the polynucleotide being in various
stages of completion;
[0332] (b) adding to the resultant population of single- or
double-stranded polynucleotides one or more single- or
double-stranded oligonucleotides, wherein said added
oligonucleotides comprise an area of identity in an area of
heterology to one or more of the single- or double-stranded
polynucleotides of the population;
[0333] (c) denaturing the resulting single- or double-stranded
oligonucleotides to produce a mixture of single-stranded
polynucleotides, optionally separating the shorter or smaller
polynucleotides into pools of polynucleotides having various
lengths and further optionally subjecting said polynucleotides to a
PCR procedure to amplify one or more oligonucleotides comprised by
at least one of said polynucleotide pools;
[0334] (d) incubating a plurality of said polynucleotides or at
least one pool of said polynucleotides with a polymerase under
conditions which result in annealing of said single-stranded
polynucleotides at regions of identity between the single-stranded
polynucleotides and thus forming of a mutagenized double-stranded
polynucleotide chain;
[0335] (e) optionally repeating steps (c) and (d);
[0336] (f) expressing at least one hybrid polypeptide from said
polynucleotide chain, or chains; and
[0337] (g) screening said at least one hybrid polypeptide for a
useful activity.
[0338] In a preferred aspect of the invention, the means for
blocking or interrupting the amplification or synthesis process is
by utilization of uv light, DNA adducts, DNA binding proteins.
[0339] In one embodiment of the invention, the DNA adducts, or
polynucleotides comprising the DNA adducts, are removed from the
polynucleotides or polynucleotide pool, such as by a process
including heating the solution comprising the DNA fragments prior
to further processing.
[0340] Having thus disclosed exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
disclosures are exemplary only and that various other alternatives,
adaptations and modifications may be made within the scope of the
present invention. Accordingly, the present invention is not
limited to the specific embodiments as illustrated herein.
[0341] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
Generation of Random Size Polynucleotides Using U.V. Induced
Photoproducts
[0342] One microgram samples of template DNA are obtained and
treated with U.V. light to cause the formation of dimers, including
TT dimers, particularly purine dimers. U.V. exposure is limited so
that only a few photoproducts are generated per gene on the
template DNA sample. Multiple samples are treated with U.V. light
for varying periods of time to obtain template DNA samples with
varying numbers of dimers from U.V. exposure.
[0343] A random priming kit which utilizes a non-proofreading
polymease (for example, Prime-It II Random Primer Labeling kit by
Stratagene Cloning Systems) is utilized to generate different size
polynucleotides by priming at random sites on templates which are
prepared by U.V. light (as described above) and extending along the
templates. The priming protocols such as described in the Prime-It
II Random Primer Labeling kit may be utilized to extend the
primers. The dimers formed by U.V. exposure serve as a roadblock
for the extension by the non-proofreading polymerase. Thus, a pool
of random size polynucleotides is present after extension with the
random primers is finished.
EXAMPLE 2
Isolation of Random Size Polynucleotides
[0344] Polynucleotides of interest which are generated according to
Example 1 are are gel isolated on a 1.5% agarose gel.
Polynucleotides in the 100-300 bp range are cut out of the gel and
3 volumes of 6 M NaI is added to the gel slice. The mixture is
incubated at 50.degree. C. for 10 minutes and 10 .mu.l of glass
milk (Bio 101) is added. The mixture is spun for 1 minute and the
supernatant is decanted. The pellet is washed with 500 .mu.l of
Column Wash (Column Wash is 50% ethanol, 10 mM Tris-HCl pH 7.5, 100
mM NaCl and 2.5 mM EDTA) and spin for 1 minute, after which the
supernatant is decanted. The washing, spinning and decanting steps
are then repeated. The glass milk pellet is resuspended in 20 .mu.l
of H.sub.2O and spun for 1 minute. DNA remains in the aqueous
phase.
EXAMPLE 3
Shuffling of Isolated Random Size 100-300bp Polynucleotides
[0345] The 100-300 bp polynucleotides obtained in Example 2 are
recombined in an annealing mixture (0.2 mM each dNTP, 2.2 mM
MgCl.sub.2, 50 mM KCl, 10 mM Tris-HCl ph 8.8, 0.1% Triton X-100,
0.3.mu.; Taq DNA polymerase, 50 .mu.l total volume) without adding
primers. A Robocycler by Stratagene was used for the annealing step
with the following program: 95.degree. C. for 30 seconds. 25-50
cycles of [95.degree. C. for 30 seconds, 50-60.degree. C.
(preferably 58.degree. C.) for 30 seconds, and 72.degree. C. for 30
seconds] and 5 minutes at 72.degree. C. Thus, the 100-300 bp
polynucleotides combine to yield double-stranded polynucleotides
having a longer sequence. After separating out the reassembled
double-stranded polynucleotides and denaturing them to form single
stranded polynucleotides, the cycling is optionally again repeated
with some samples utilizing the single strands as template and
primer DNA and other samples utilizing random primers in addition
to the single strands.
EXAMPLE 4
Screening of Polvpeptides from Shuffled Polynucleotides
[0346] The polynucleotides of Example 3 are separated and
polypeptides are expressed therefrom. The original template DNA is
utilized as a comparative control by obtaining comparative
polypeptides therefrom. The polypeptides obtained from the shuffled
polynucleotides of Example 3 are screened for the activity of the
polypeptides obtained from the original template and compared with
the activity levels of the control. The shuffled polynucleotides
coding for interesting polypeptides discovered during screening are
compared further for secondary desirable traits. Some shuffled
polynucleotides corresponding to less interesting screened
polypeptides are subjected to reshuffling.
EXAMPLE 5
Directed Evolution an Enzyme by Saturation Mutagenesis
[0347] Site-Saturation Mutagenesis: To accomplish site-saturation
mutagenesis every residue (316) of a dehalogenase enzyme was
converted into all 20 amino acids by site directed mutagenesis
using 32-fold degenerate oligonucleotide primers, as follows:
[0348] 1. A culture of the dehalogenase expression construct was
grown and a preparation of the plasmid was made
[0349] 2. Primers were made to randomize each codon--they have the
common structure X.sub.20NN(G/T)X.sub.20
[0350] 3. A reaction mix of 25 ul was prepared containing .about.50
ng of plasmid template, 125 ng of each primer, 1X native P fu
buffer, 200 uM each dNTP and 2.5 U native Pfu DNA polymerase
[0351] 4. The reaction was cycled in a Robo96 Gradient Cycler as
follows:
[0352] Initial denaturation at 95.degree. C. for 1 min
[0353] 20 cycles of 95.degree. C. for 45 sec, 53.degree. C. for 1
min and 72.degree. C. for 11 min
[0354] Final elongation step of 72.degree. C. for 10 min
[0355] 5. The reaction mix was digested with 10 U of DpnI at
37.degree. C. for 1 hour to digest the methylated template DNA
[0356] 6. Two ul of the reaction mix were used to transform 50 ul
of XL1-Blue MRF' cells and the entire transformation mix was plated
on a large LB-Amp-Met plate yielding 200-1000 colonies
[0357] 7. Individual colonies were toothpicked into the wells of
96-well microtiter plates containing LB-Amp-IPTG and grown
overnight
[0358] 8. The clones on these plates were assayed the following
day
[0359] Screening: Approximately 200 clones of mutants for each
position were grown in liquid media (384 well microtiter plates)
and screened as follows:
[0360] 1. Overnight cultures in 384-well plates were centrifuged
and the media removed. To each well was added 0.06 mL 1 mM
Tris/SO.sub.4.sup.2- pH 7.8.
[0361] 2. Made 2 assay plates from each parent growth plate
consisting of 0.02 mL cell suspension.
[0362] 3. One assay plate was placed at room temperature and the
other at elevated temperature (initial screen used 55.degree. C.)
for a period of time (initially 30 minutes).
[0363] 4. Afier the prescribed time 0.08 mL room temperature
substrate (TCP saturated 1 mM Tris/SO.sub.4.sup.2- pH 7.8 with 1.5
mM NaN.sub.3 and 0.1 mM bromothymol blue) was added to each
well.
[0364] 5. Measurements at 620 nm were taken at various time points
to generate a progress curve for each well.
[0365] 6. Data were analyzed and the kinetics of the cells heated
to those not heated were compared. Each plate contained 1-2 columns
(24 wells) of unmutated 20F12 controls.
[0366] 7. Wells that appeared to have improved stability were
re-grown and tested under the same conditions.
[0367] Following this procedure nine single site mutations appeared
to confer increased thermal stability on the enzyme. Sequence
analysis was performed to determine of the exact amino acid changes
at each position that were specifically responsible for the
improvement. In sum, the improvement was conferred at 7 sites by
one amino acid change alone, at an eighth site by each of two amino
acid changes, and at a ninth site by each of three amino acid
changes. Several mutants were then made each having a plurality of
these nine beneficial site mutations in combination; of these two
mutants proved superior to all the other mutants, including those
with single point mutations.
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Sequence CWU 1
1
2 1 5 PRT Artificial Sequence Description of Artificial Sequence
Synthetic spacer peptide 1 Gly Gly Gly Gly Ser 1 5 2 15 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
linker peptide 2 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15
* * * * *