U.S. patent application number 10/458523 was filed with the patent office on 2003-11-20 for altered thermostability of enzymes.
Invention is credited to Short, Jay M..
Application Number | 20030215883 10/458523 |
Document ID | / |
Family ID | 41527787 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215883 |
Kind Code |
A1 |
Short, Jay M. |
November 20, 2003 |
Altered thermostability of enzymes
Abstract
Provided is a method of screening gene libraries derived from a
mixed population of organisms for a bioactivity or biomolecule of
interest. The mixed population of organisms can be a cultured
population or an uncultured population from, for example, the
environment. Also provided are methods of screening isolates or
enriched populations of organisms, which isolates include a
population that is spatially, temporally, or hierarchical, for
example, of a particular species, genus, family, or class of
organisms. Identified clones containing a biomolecule or
bioactivity of interest can be further variegated or the DNA
contained in the clone can be variegated to create novel
biomolecules or bioactivities of interest.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
HALE AND DORR LLP
300 PARK AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
41527787 |
Appl. No.: |
10/458523 |
Filed: |
June 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10458523 |
Jun 9, 2003 |
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09714780 |
Nov 15, 2000 |
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10458523 |
Jun 9, 2003 |
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09663620 |
Sep 15, 2000 |
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09663620 |
Sep 15, 2000 |
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09535754 |
Mar 27, 2000 |
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6361974 |
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09663620 |
Sep 15, 2000 |
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09375605 |
Aug 17, 1999 |
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09375605 |
Aug 17, 1999 |
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08651568 |
May 22, 1996 |
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5939250 |
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60008316 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/7.1 ;
435/189; 435/193; 435/194; 435/196; 435/198; 435/69.1; 702/19 |
Current CPC
Class: |
C12N 15/102 20130101;
C12Y 302/01021 20130101; C12N 9/00 20130101; C12N 15/1093 20130101;
C12N 9/16 20130101; C12N 15/1027 20130101; C12N 15/1034 20130101;
C12N 9/2445 20130101 |
Class at
Publication: |
435/7.1 ;
435/69.1; 435/189; 435/193; 435/194; 435/196; 435/198; 702/19 |
International
Class: |
G01N 033/53; G06F
019/00; G01N 033/48; G01N 033/50; C12N 009/02; C12N 009/10; C12N
009/12; C12N 009/16; C12N 009/20 |
Claims
What is claimed is:
1. A method for obtaining a bioactive protein having a
thermostability that is altered as compared to that of the
corresponding wild-type protein, comprising: a) variegating a
nucleic acid sequence encoding the wild-type protein; and b)
comparing the bioactivity after variegation with the bioactivity of
the wild-type protein, wherein a difference in the bioactivity is
indicative of an effect of sequence variegation, thereby providing
the a bioactive protein having a thermostability that is altered as
compared to that of the corresponding wild-type protein.
2. The method of claim 1, further comprising comparing the
variegated nucleic acid sequence of interest to the non-variegated
nucleic acid sequence of (c), thereby identifying the nucleotide
sequence variegation.
3. The method of claim 2, wherein the comparison is performed using
a sequence comparison algorithm.
4. The method of claim 1, wherein the bioactivity is an enzymatic
activity.
5. The method of claim 4, wherein the enzymatic activity is
provided by an enzyme selected from the group consisting of
lipases, esterases, proteases, glycosidases, glycosyl transferases,
phosphatases, dehydrogenases, kinases, mono- and dioxygenases,
haloperoxidases, lignin peroxidases, diarylpropane peroxidases,
epozide hydrolases, nitrile hydratases, nitrilases, transaminases,
amidases, and acylases.
6. The method of claim 1, wherein the bioactivity is identified
from an expression library.
7. The method of claim 6, wherein the library contains DNA obtained
from an environmental sample.
8. The method of claim 6, wherein the library contains nucleic acid
obtained from extremophiles.
9. The method of claim 8, wherein the extremophiles are
thermophiles.
10. The method of claim 8, wherein the extremeophiles are selected
from the group consisting of hyperthermophiles, psychrophiles,
halophiles, psychrotrophs, alkalophiles, and acidophiles.
11. The method of claim 6, wherein the bioactivity is identified by
screening comprising contacting a clone with a substrate labeled
with a detectable molecule wherein interaction of the substrate
with the bioactivity contained in the clone produces a detectable
signal.
12. The method of claim 11, wherein the substrate is a bioactive
substrate.
13. The method of claim 11, wherein the bioactive substrate
comprises C12FDG.
14. The method of claim 11, wherein the substrate comprises a first
test protein linked to a DNA binding moiety and a second test
protein linked to a transcriptional activation moiety, wherein
modulation of the interaction of the first test protein linked to
the DNA binding moiety with the second test protein linked to the
transcription activation moiety results in a change in the
expression of a detectable protein.
15. The method of claim 1, further comprising, prior to (b),
obtaining nucleic acids from the clone containing the specified
bioactivity or biomolecule.
16. The method of claim 15, wherein obtaining the nucleic acids
contained in the clone comprises contacting the clone with a
complementary nucleic acid, or fragment thereof, thereby allowing
hybridization of the clone nucleic acids with the complementary
nucleic acid and isolation thereof.
17. The method of claim 16, wherein the complementary nucleic acid
or fragment thereof comprises a solid phase bound hybridization
probe.
18. The method of claim 1, wherein the nucleic acid sequence is
variegated by a method selected from the group consisting of
error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,
assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis, exponential ensemble
mutagenesis, site-specific mutagenesis, ligation reassembly, GSSM
and any combination thereof.
19. The method of claim 1, wherein the nucleic acid sequence is
variegated by error-prone PCR.
20. The method of claim 1, wherein the nucleic acid sequence is
variegated by shuffling.
21. The method of claim 1, wherein the nucleic acid sequence is
variegated by oligonucleotide-directed mutagenesis.
22. The method of claim 1, wherein the nucleic acid sequence is
variegated by assembly PCR.
23. The method of claim 1, wherein the nucleic acid sequence is
variegated by sexual PCR mutagenesis.
24. The method of claim 1, wherein the nucleic acid sequence is
variegated by in vivo mutagenesis.
25. The method of claim 1, wherein the nucleic acid sequence is
variegated by cassette mutagenesis.
26. The method of claim 1, wherein the nucleic acid sequence is
variegated by recursive ensemble mutagenesis.
27. The method of claim 1, wherein the nucleic acid sequence is
variegated by exponential ensemble mutagenesis.
28. The method of claim 1, wherein the nucleic acid sequence is
variegated by site-specific mutagenesis.
29. The method of claim 6, comprising screening a clone of the
library for a further specified protein or enzymatic activity,
prior to variegating the nucleic acids.
30. The method of claim 6, wherein the library is generated in a
prokaryotic cell.
31. The method of claim 6, wherein the library is generated in a
Streptomyces sp.
32. The method of claim 31, wherein the Streptomyces is
Streptomyces venezuelae.
33. The method of claim 30, wherein the prokaryotic cell is gram
negative.
34. The method of claim 30, wherein the prokaryotic cell is a
Bacillus sp.
35. The method of claim 30, wherein the prokaryotic cell is a
Pseudomonas sp.
36. The method of claim 6, wherein the library is screened by
contacting or encapsulating a clone of the library with bioactive
substrate, wherein a bioactivity or biomolecule produced by the
clone is detectable by a difference in the substrate prior to
contacting with the clone as compared to after contacting.
37. The method of claim 6, wherein the library is normalized before
screening the library.
38. The method of claim 1, wherein the bioactivity is a gene
cluster or fragment thereof.
39. The method of claim 1, wherein the bioactivity is a polypeptide
in a metabolic pathway.
40. The method of claim 1, wherein the thermostability is lower in
the variegated protein as compared to the wild-type protein.
41. The method of claim 1, wherein the thermostability is higher in
the variegated protein as compared to the wild-type protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/663,620, Sep. 15, 2000, which is a continuation-in-part of
U.S. application Ser. No. 09/535,754, filed Mar. 27, 2000 and U.S.
application Ser. No. 09/375,605, filed Aug. 17, 1999, which is a
continuation of U.S. application Ser. No. 08/651,568, filed May 5,
1996, issued as U.S. Pat. No. 5,939,250, which claims priority
under 35 U.S.C. .sctn.119(e) to U.S. Provisional Application Serial
No. 60/008,316, filed Dec. 7, 1995. All of the disclosures of which
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to screening and
identification of bioactive molecules and more specifically to the
production and screening of gene libraries generated from nucleic
acid isolated from more than one organism for bioactive molecules
or bioactivities.
BACKGROUND
[0003] The majority of bioactive compounds currently in use are
derived from soil microorganisms. Many microbes inhabiting soils
and other complex ecological communities produce a variety of
compounds that increase their ability to survive and proliferate.
These compounds are generally thought to be nonessential for growth
of the organism and are synthesized with the aid of genes involved
in intermediary metabolism. Such secondary metabolites that
influence the growth or survival of other organisms are known as
.quadrature.bioactive.quadrature. compounds and serve as key
components of the chemical defense arsenal of both micro- and
macroorganisms. Humans have exploited these compounds for use as
antibiotics, antiinfectives and other bioactive compounds with
activity against a broad range of prokaryotic and eukaryotic
pathogens (Barnes et al., Proc. Nat. Acad. Sci. U.S.A., 91,
1994).
[0004] Despite the seemingly large number of available bioactive
compounds, it is clear that one of the greatest challenges facing
modern biomedical science is the proliferation of antibiotic
resistant pathogens. Because of their short generation time and
ability to readily exchange genetic information, pathogenic
microbes have rapidly evolved and disseminated resistance
mechanisms against virtually all classes of antibiotic compounds.
For example, there are virulent strains of the human pathogens
Staphylococcus and Streptococcus that can now be treated with but a
single antibiotic, vancomycin, and resistance to this compound will
require only the transfer of a single gene, vanA, from resistant
Enterococcus species for this to occur. (Bateson et al., System.
Appl. Microbiol, 12, 1989). When this crucial need for novel
antibacterial compounds is superimposed on the growing demand for
enzyme inhibitors, immunosuppressants and anti-cancer agents it
becomes readily apparent why pharmaceutical companies have stepped
up their screening of microbial samples for bioactive
compounds.
[0005] The approach currently used to screen microbes for new
bioactive compounds has been largely unchanged since the inception
of the field. New isolates of bacteria, particularly gram positive
strains from soil environments, are collected and their metabolites
tested for pharmacological activity.
[0006] There is still tremendous biodiversity that remains untapped
as the source of lead compounds. However, the currently available
methods for screening and producing lead compounds cannot be
applied efficiently to these under-explored resources. For
instance, it is estimated that at least 99% of marine bacteria
species do not survive on laboratory media, and commercially
available fermentation equipment is not optimal for use in the
conditions under which these species will grow, hence these
organisms are difficult or impossible to culture for screening or
re-supply. Recollection, growth, strain improvement, media
improvement and scale-up production of the drug-producing organisms
often pose problems for synthesis and development of lead
compounds. Furthermore, the need for the interaction of specific
organisms to synthesize some compounds makes their use in discovery
extremely difficult. New methods to harness the genetic resources
and chemical diversity of these untapped sources of compounds for
use in drug discovery are very valuable.
[0007] A central core of modern biology is that genetic information
resides in a nucleic acid genome, and that the information embodied
in such a genome (i.e., the genotype) directs cell function. This
occurs through the expression of various genes in the genome of an
organism and regulation of the expression of such genes. The
expression of genes in a cell or organism defines the cell or
organism's physical characteristics (i.e., its phenotype). This is
accomplished through the translation of genes into proteins.
Determining the biological activity of a protein obtained from an
environmental sample can provide valuable information about the
role of proteins in the environments. In addition, such information
can help in the development of biologics, diagnostics,
therapeutics, and compositions for industrial applications.
[0008] Accordingly, the present invention provides methods to
access this untapped biodiversity and to rapidly screen for
sequences and activities of interest utilizing recombinant DNA
technology. This invention combines the benefits associated with
the ability to rapidly screen natural compounds with the
flexibility and reproducibility afforded with working with the
genetic material of organisms.
SUMMARY OF THE INVENTION
[0009] The present invention provides rapid screening of samples
for bioactivities or biomolecules of interest. Samples can be
derived from a wide range of sources and include, for example,
environmental libraries, samples containing more than one organisms
(e.g., mixed populations of organisms), samples from unculturable
organisms, deep sea vents and the like. As described herein, such
samples provide a rich source of untapped molecules useful in
biologics, therapeutics and industrial application, which prior to
the present invention required laborious and time consuming methods
for characterization and identification or were unable to be
identified or characterized.
[0010] In one embodiment the invention provides a method for
obtaining a bioactivity or a biomolecule of interest by screening a
library of clones generated from nucleic acids from a mixed
population of cells, for a specified bioactivity or biomolecule,
variegating a nucleic acid sequence contained in a clone having the
specified bioactivity or biomolecule; and comparing the variegated
bioactivity or biomolecule with the specified bioactivity or
biomolecule wherein a difference in the bioactivity or biomolecule
is indicative of an effect of sequence variegation, thereby
providing the bioactivity or biomolecule of interest.
[0011] In another embodiment, the invention provides a method for
identifying a bioactivity or a biomolecule of interest by screening
a library of clones generated from pooled nucleic acids obtained
from a plurality of isolates for a specified bioactivity or
biomolecule; and identifying a clone which contains the specified
bioactivity or biomolecule.
[0012] In yet another embodiment, the invention provides a method
for identifying a bioactivity or a biomolecule of interest. The
method includes screening a library of clones generated from pooled
nucleic acids obtained from a plurality of isolates for a specified
bioactivity or biomolecule, variegating a nucleic acid sequence
contained in a clone having the specified bioactivity or
biomolecule, and comparing the variegated bioactivity or
biomolecule with the specified bioactivity or biomolecule wherein a
difference in the bioactivity or biomolecule is indicative of an
effect of introducing at least one sequence variegation, thereby
providing the bioactivity or biomolecule of interest.
[0013] In another embodiment, the invention provides a method for
identifying a bioactivity or a biomolecule of interest, wherein the
method includes screening a library of clones generated from
pooling individual gene libraries generated from the nucleic acids
obtained from each of a plurality of isolates for a specified
bioactivity or biomolecule and identifying a clone which contains
the specified bioactivity or biomolecule.
[0014] In another embodiment, the invention provides a method for
identifying a bioactivity or a biomolecule of interest by screening
a library for a specified bioactivity or biomolecule wherein the
library is generated from pooling individual gene libraries
generated from the nucleic acids obtained from each of a plurality
of isolates, variegating a nucleic acid sequence contained in a
clone having the specified bioactivity or biomolecule, and
comparing the variegated bioactivity or biomolecule with the
specified bioactivity or biomolecule wherein a difference in the
bioactivity or biomolecule is indicative of an effect of
introducing at least one sequence variegation, thereby providing
the bioactivity or biomolecule of interest.
[0015] In yet another embodiment, the invention provides a method
of identifying a bioactivity or biomolecule of interest, including
screening a library of clones generated from the nucleic acids from
an enriched population of organisms for a specified bioactivity or
biomolecule and identifying a clone containing the specified
bioactivity or biomolecule.
[0016] In yet another embodiment, the invention provides a method
of identifying a bioactivity or biomolecule of interest by
screening a library of clones generated from nucleic acids from an
enriched population of organisms for a specified bioactivity or
biomolecule, variegating a nucleic acid sequence contained in a
clone having the specified bioactivity or biomolecule, and
comparing the variegated bioactivity or biomolecule with the
specified bioactivity or biomolecule wherein a difference in the
bioactivity or biomolecule is indicative of an effect of
introducing at least one sequence variegation, thereby providing
the bioactivity or biomolecule of interest.
[0017] In another embodiment, the invention provides a method for
identifying a bioactivity or a biomolecule of interest. The
bioactivity or biomolecule of interest is identified by incubating
nucleic acids from a mixed population of organisms with at least
one oligonucleotide probe having a detectable molecule and at least
a portion of a nucleic acid sequence encoding a molecule of
interest under conditions to allow interaction of complementary
sequences, identifying nucleic acid sequences having a complement
to the oligonucleotide probe using an analyzer that detects the
detectable molecule. A library is then generated from the
identified nucleic acid sequences and the library is screened for a
specified bioactivity or biomolecule. Nucleic acid sequence
contained in a clone having the specified bioactivity or
biomolecule is variegated and the variegated bioactivity or
biomolecule compared with the specified bioactivity or biomolecule
wherein a difference in the bioactivity or biomolecule is
indicative of an effect of introducing at least one sequence
variation, thereby providing the bioactivity or biomolecule of
interest
[0018] In another embodiment, the invention provides a method for
identifying a bioactivity or a biomolecule of interest by
co-encapsulating in a microenvironment nucleic acids obtained from
a mixed population of organisms, with at least one oligonucleotide
probe having a detectable molecule and at least a portion of a
nucleic acid sequence encoding a molecule of interest under such
conditions and for such time as to allow interaction of
complementary sequences, identifying encapsulated nucleic acids
containing a complement to the oligonucleotide probe encoding the
molecule of interest by separating the encapsulated nucleic acids
with an analyzer that detects the detectable molecule, generating a
library from the separated encapsulated nucleic acids, screening
the library for a specified bioactivity or biomolecule, variegating
a nucleic acid sequence contained in a clone having the specified
bioactivity or biomolecule, and comparing the variegated
bioactivity or biomolecule with the specified bioactivity or
biomolecule wherein a difference in the bioactivity or biomolecule
is indicative of an effect of introducing at least one sequence
variation, thereby providing the bioactivity or biomolecule of
interest.
[0019] In yet another embodiment, the invention provides a method
including co-encapsulating in a microenvironment nucleic acids
obtained from an isolate of a mixed population of organisms, with
at least one oligonucleotide probe having a detectable marker and
at least a portion of a polynucleotide sequence encoding a molecule
having a bioactivity of interest under conditions and for such time
as to allow interaction of complementary sequences, identifying
encapsulated nucleic acids containing a complement to the
oligonucleotide probe encoding the molecule of interest by
separating the encapsulated nucleic acids with an analyzer that
detects the detectable marker, generating a library from the
separated encapsulated nucleic acids, screening the library for a
specified bioactivity or biomolecule, variegating a nucleic acid
sequence contained in a clone having the specified bioactivity or
biomolecule, and comparing the variegated bioactivity or
biomolecule with the specified bioactivity or biomolecule wherein a
difference in the bioactivity or biomolecule is indicative of an
effect of introducing at least one sequence variation, thereby
providing the bioactivity or biomolecule of interest.
[0020] In another embodiment, the invention provides a method for
obtaining a bioactivity or a biomolecule of interest by
co-encapsulating in a microenvironment nucleic acids obtained from
one or more isolates of a mixed population of organisms, with at
least one oligonucleotide probe having a detectable marker and at
least a portion of a polynucleotide sequence encoding a molecule
having a bioactivity of interest under such conditions and for such
time as to allow interaction of complementary sequences,
identifying encapsulated nucleic acids containing a complement to
the oligonucleotide probe encoding the molecule of interest by
separating the encapsulated nucleic acids with an analyzer that
detects the detectable marker, generating a library from the
separated encapsulated nucleic acids, screening the library for a
specified bioactivity or biomolecule, variegating a nucleic acid
sequence contained in a clone having the specified bioactivity or
biomolecule, and comparing the variegated bioactivity or
biomolecule with the specified bioactivity or biomolecule wherein a
difference in the bioactivity or biomolecule is indicative of an
effect of introducing at least one sequence variation, thereby
providing the bioactivity or biomolecule of interest.
[0021] In yet another embodiment, the invention provides a method
for identifying a bioactivity or a biomolecule of interest. The
method includes co-encapsulating in a microenvironment nucleic
acids obtained from a mixture of isolates of a mixed population of
organisms, with at least one oligonucleotide probe having a
detectable marker and at least a portion of a polynucleotide
sequence encoding a molecule having a bioactivity of interest under
such conditions and for such time as to allow interaction of
complementary sequences, identifying encapsulated nucleic acids
containing a complement to the oligonucleotide probe encoding the
molecule of interest by separating the encapsulated nucleic acids
with an analyzer that detects the detectable marker, generating a
library from the separated encapsulated nucleic acids, screening
the library for a specified bioactivity or biomolecule, variegating
the a nucleic acid sequence contained in a clone having the
specified bioactivity or biomolecule, and comparing the variegated
bioactivity or biomolecule with the specified bioactivity or
biomolecule wherein a difference in the bioactivity or biomolecule
is indicative of an effect of introducing at least one sequence
variation, thereby providing the bioactivity or biomolecule of
interest.
[0022] These and other aspects of the present invention will be
apparent to those skilled in the art from the teachings herein.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides rapid screening of libraries
derived from more than one organism, such as a mixed population of
organisms from, for example, an environmental sample or an
uncultivated population of organisms or a cultivated population of
organisms.
[0024] In one embodiment, gene libraries are generated by obtaining
nucleic acids from a mixed population of organisms and cloning the
nucleic acids into a suitable vector for transforming a plurality
of clones to generate a gene library. The gene library thus
contains gene or gene fragments present in organisms of the mixed
population. The gene library can be an expression library, in which
case the library can be screened for an expressed polypeptide
having a desired activity. Alternatively, the gene library can be
screened for sequences of interest by, for example, PCR or
hybridization screening.
[0025] In one embodiment, nucleic acids from isolates of a sample
containing a mixed population of organism are pooled and the pooled
nucleic acids are used to generate a gene library.
[0026] By "isolates" is meant that a particular species, genus,
family, order, or class of organisms is obtained or derived from a
sample having more than one organism or from a mixed population of
organisms. Nucleic acids from these isolated populations can then
be used to generate a gene library. Isolates can be obtained from
by selectively filtering or culturing a sample containing more than
one organism or a mixed population of organisms. For example,
isolates of bacteria can be obtained by filtering the sample
through a filter which excludes organisms based on size or by
culturing the sample on media that allows from selective growth or
selective inhibition of certain populations of organisms.
[0027] An "enriched population" is a population of organisms
wherein the percentage of organisms belonging to a particular
species, genus, family, order or class of organisms is increased
with respect to the population as a whole. For example, selective
growth or inhibition media can increase the overall number of
organisms. One can enrich for prokaryotic organisms with respect to
the total number of organisms in the population. Similarly, a
particular species, genus, family, order or class of organisms can
be enriched by growing a mixed population on a selective media that
inhibits or promotes the growth of a subpopulation within the mixed
population.
[0028] In another embodiment, nucleic acids from a plurality (e.g.,
two or more) of isolates from a mixed population of organisms are
used to generate a plurality of gene libraries containing a
plurality of clones, and the gene libraries from at least two
isolates are then pooled to obtain a "pooled isolate library."
[0029] Once gene libraries are generated, the clones are screened
to detect a bioactivity (e.g., an enzymatic activity, secondary
messenger activity, binding activity, transcriptional activity and
the like) or a biomolecule of interest (e.g., a nucleic acid
sequence, a peptide, a polypeptide, a lipid or other small
molecule, and the like). Such screening techniques include, for
example, contacting a clone, clonal population, or population of
nucleic acid sequences with a substrate or substrates having a
detectable molecule that provides a detectable signal upon
interaction with the bioactivity or biomolecule of interest. The
substrate can be an enzymatic substrate, a bioactive molecule, an
oligonucleotide, and the like.
[0030] In one embodiment, gene libraries are generated, clones are
either exposed to a chromagenic or fluorogenic substrate or
substrate(s) of interest, or hybridized to a labeled probe (e.g.,
an oligonucleotide having a detectable molecule) having a sequence
corresponding to a sequence of interest and positive clones are
identified by a detectable signal (e.g., fluorescence
emission).
[0031] In one embodiment, expression libraries generated from a
mixed population of organisms are screened for an activity of
interest. Specifically, expression libraries are generated, clones
are exposed to the substrate or substrate(s) of interest, and
positive clone are identified and isolated. The present invention
does not require cells to survive. The cells only need to be viable
long enough to produce the molecule to be detected, and can
thereafter be either viable or nonviable cells, so long as the
expressed biomolecule (e.g., an enzyme) remains active.
[0032] In certain embodiment, the invention provides an approach
that combines direct cloning of genes encoding novel or desired
bioactivities from environmental samples with a high-throughput
screening system designed for the rapid discovery of new molecules,
for example, enzymes. The approach is based on the construction of
environmental "expression libraries" which can represent the
collective genomes of numerous naturally occurring microorganisms
archived in cloning vectors that can be propagated in E. coli or
other suitable host cells. Because the cloned DNA can be initially
extracted directly from environmental samples or from isolates of
the environmental samples, the libraries are not limited to the
small fraction of prokaryotes that can be grown in pure culture.
Additionally, a normalization of the environmental DNA present in
these samples could allows a more equal representation of the DNA
from all of the species present in a sample. Normalization
techniques (described below) can dramatically increase the
efficiency of finding interesting genes from minor constituents of
the sample that may be under-represented by several orders of
magnitude compared to the dominant species in the sample.
Normilization can occur in any of the foregoing embodiments
following obtaining nucleic acids from the sample or
isolate(s).
[0033] In another embodiment, the invention provides a
high-throughput capillary array system for screening that allows
one to assess an enormous number of clones to identify and recover
cells encoding useful enzymes, as well as other biomolecules (e.g.,
ligands). In particular, the capillary array-based techniques
described herein can be used to screen, identify and recover
proteins having a desired bioactivity or other ligands having a
desired binding affinity. For example, binding assays may be
conducted by using an appropriate substrate or other marker that
emits a detectable signal upon the occurrence of the desired
binding event.
[0034] In addition, fluorescence activated cell sorting can be used
to screen and isolate clones having an activity or sequence of
interest. Previously, FACS machines have been employed in the
studies focused on the analyses of eukaryotic and prokaryotic cell
lines and cell culture processes. FACS has also been utilized to
monitor production of foreign proteins in both eukaryotes and
prokaryotes to study, for example, differential gene expression,
and the like. The detection and counting capabilities of the FACS
system have been applied in these examples. However, FACS has never
previously been employed in a discovery process to screen for and
recover bioactivities in prokaryotes. Furthermore, the present
invention does not require cells to survive, as do previously
described technologies, since the desired nucleic acid (recombinant
clones) can be obtained from alive or dead cells. The cells only
need to be viable long enough to produce the compound to be
detected, and can thereafter be either viable or non-viable cells
so long as the expressed biomolecule remains active. The present
invention also solves problems that would have been associated with
detection and sorting of E. coli expressing recombinant enzymes,
and recovering encoding nucleic acids. Additionally, the present
invention includes within its embodiments any apparatus capable of
detecting fluorescent wavelengths associated with biological
material, such apparati are defined herein as fluorescent analyzers
(one example of which is a FACS apparatus).
[0035] In some instances it is desirable to identify nucleic acid
sequences from a mixed population of organisms, isolates, or
enriched populations. In this embodiment, it is not necessary to
express gene products. Nucleic acid sequences of interest can be
identified or "biopanned" by contacting a clone, device (e.g. a
gene chip), filter, or nucleic acid sample with a probe labeled
with a detectable molecule. The probe will typically have a
sequence that is substantially identical to the nucleic acid
sequence of interest, Alternatively, the probe will be a fragment
or full length nucleic acid sequence encoding a polypeptide of
interest. The prove and nucleic acids are incubated under
conditions and for such time as to allow the probe and a
substantially complementary sequence to hybridize. Hybridization
stringency will vary depending on, for example, the length and GC
content of the probe. Such factors can be determined empirically
(See, for example, Sambrook et al., Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989, and Current Protocols in Molecular Biology, M. Ausubel et
al., eds., (Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc., most
recent Supplement)). Once hybridized the complementary sequence can
be PCR amplified, identified by hybridization techniques (e.g.,
exposing the probe and nucleic acid mixture to a film), or
detecting the nucleic acid using a chip.
[0036] Prior to the present invention, the evaluation of complex
gene libraries or environmental expression libraries was rate
limiting. The present invention allows the rapid screening of
complex environmental libraries, containing, for example, genomic
sequences from thousands of different organisms or subsets and
isolates thereof. The benefits of the present invention can be
seen, for example, in screening a complex environmental sample.
Screening of a complex sample previously required one to use labor
intensive methods to screen several million clones to cover the
genomic biodiversity. The invention represents an extremely
high-throughput screening method which allows one to assess this
enormous number of clones. The method disclosed allows the
screening anywhere from about 30 million to about 200 million
clones per hour for a desired nucleic acid sequence, biological
activity, or biomolecule of interest. This allows the thorough
screening of environmental libraries for clones expressing novel
bioactivities or biomolecules.
[0037] Once a sequence or bioactivity of interest is identified
(e.g., an enzyme of interest) the sequence or polynucleotide
encoding the bioactivity of interest can be evolved, mutated or
derived to modify the amino acid sequence to provide, for example,
modified activities such as increased thermostability, specificity
or activity.
[0038] An "amino acid" is a molecule having the structure wherein a
central carbon atom (the I-carbon atom) is linked to a hydrogen
atom, a carboxylic acid group (the carbon atom of which is referred
to herein as a "carboxyl carbon atom"), an amino group (the
nitrogen atom of which is referred to herein as an "amino nitrogen
atom"), and a side chain group, R. When incorporated into a
peptide, polypeptide, or protein, an amino acid loses one or more
atoms of its amino acid carboxylic groups in the dehydration
reaction that links one amino acid to another. As a result, when
incorporated into a protein, an amino acid is referred to as an
"amino acid residue."
[0039] "Protein" or "polypeptide" refers to any polymer of two or
more individual amino acids (whether or not naturally occurring)
linked via a peptide bond, and occurs when the carboxyl carbon atom
of the carboxylic acid group bonded to the I-carbon of one amino
acid (or amino acid residue) becomes covalently bound to the amino
nitrogen atom of amino group bonded to the I-carbon of an adjacent
amino acid. The term "protein" is understood to include the terms
"polypeptide" and "peptide" (which, at times may be used
interchangeably herein) within its meaning. In addition, proteins
comprising multiple polypeptide subunits (e.g., DNA polymerase III,
RNA polymerase II) or other components (for example, an RNA
molecule, as occurs in telomerase) will also be understood to be
included within the meaning of "protein" as used herein. Similarly,
fragments of proteins and polypeptides are also within the scope of
the invention and may be referred to herein as "proteins."
[0040] A particular amino acid sequence of a given protein (i.e.,
the polypeptide's "primary structure," when written from the
amino-terminus to carboxy-terminus) is determined by the nucleotide
sequence of the coding portion of a mRNA, which is in turn
specified by genetic information, typically genomic DNA (including
organelle DNA, e.g., mitochondrial or chloroplast DNA). Thus,
determining the sequence of a gene assists in predicting the
primary sequence of a corresponding polypeptide and more particular
the role or activity of the polypeptide or proteins encoded by that
gene or polynucleotide sequence.
[0041] The term "isolated" or "purified" when referring to a
nucleic acid sequence or a polypeptide sequence, respectively,
means altered "by the hand of man" from its natural state; i.e., if
it occurs in nature, it has been changed or removed from its
original environment, or both. For example, a naturally occurring
polynucleotide or a polypeptide naturally present in a living
animal, a biological sample or an environmental sample in its
natural state is not "isolated" or "purified", but the same
polynucleotide or polypeptide separated from the coexisting
materials of its natural state is "isolated" or "purified", as the
term is employed herein. Such polynucleotides, when introduced into
host cells in culture or in whole organisms, still would be
isolated, as the term is used herein, because they would not be in
their naturally occurring form or environment. Similarly, the
polynucleotides and polypeptides may occur in a composition, such
as a media formulation (solutions for introduction of
polynucleotides or polypeptides, for example, into cells or
compositions or solutions for chemical or enzymatic reactions).
[0042] "Polynucleotide" or "nucleic acid sequence" refers to a
polymeric form of nucleotides. In some instances a polynucleotide
refers to a sequence that is not immediately contiguous with either
of the coding sequences with which it is immediately contiguous
(one on the 5' end and one on the 3' end) in the naturally
occurring genome of the organism from which it is derived. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA)
independent of other sequences. The nucleotides of the invention
can be ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. A polynucleotides as used herein refers to,
among others, single-and double-stranded DNA, DNA that is a mixture
of single- and double-stranded regions, single- and double-stranded
RNA, and RNA that is mixture of single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or a mixture of
single- and double-stranded regions.
[0043] In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide. The term polynucleotide encompasses
genomic DNA or RNA (depending upon the organism, i.e., RNA genome
of viruses), as well as mRNA encoded by the genomic DNA, and
cDNA.
[0044] As mentioned above, there is currently a need in the
biotechnology and chemical industry for molecules that can
optimally carry out biological or chemical processes (e.g.,
enyzmes). For example, molecules and compounds that are utilized in
both established and emerging chemical, pharmaceutical, textile,
food and feed, and detergent markets must meet stringent economical
and environmental standards. The synthesis of polymers,
pharmaceuticals, natural products and agrochemicals is often
hampered by expensive processes which produce harmful byproducts
and which suffer from poor or inefficient catalysis. Enzymes, for
example, have a number of remarkable advantages which can overcome
these problems in catalysis: they act on single functional groups,
they distinguish between similar functional groups on a single
molecule, and they distinguish between enantiomers. Moreover, they
are biodegradable and function at very low mole fractions in
reaction mixtures. Because of their chemo-, regio- and
stereospecificity, enzymes present a unique opportunity to
optimally achieve desired selective transformations. These are
often extremely difficult to duplicate chemically, especially in
single-step reactions. The elimination of the need for protection
groups, selectivity, the ability to carry out multi-step
transformations in a single reaction vessel, along with the
concomitant reduction in environmental burden, has led to the
increased demand for enzymes in chemical and pharmaceutical
industries. Enzyme-based processes have been gradually replacing
many conventional chemical-based methods. A current limitation to
more widespread industrial use is primarily due to the relatively
small number of commercially available enzymes. Only .about.300
enzymes (excluding DNA modifying enzymes) are at present
commercially available from the >3000 non DNA-modifying enzyme
activities thus far described.
[0045] The use of enzymes for technological applications also may
require performance under demanding industrial conditions. This
includes activities in environments or on substrates for which the
currently known arsenal of enzymes was not evolutionarily selected.
However, the natural environment provides extreme conditions
including, for example, extremes in temperature and pH. A number of
organisms have adapted to these conditions due in part to selection
for polypeptides than can withstand these extremes.
[0046] Enzymes have evolved by selective pressure to perform very
specific biological functions within the milieu of a living
organism, under conditions of temperature, pH and salt
concentration. For the most part, the non-DNA modifying enzyme
activities thus far identified have been isolated from mesophilic
organisms, which represent a very small fraction of the available
phylogenetic diversity. The dynamic field of biocatalysis takes on
a new dimension with the help of enzymes isolated from
microorganisms that thrive in extreme environments. For example,
such enzymes must 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. Environmental samples obtained, for example, from
extreme conditions containing organisms, polynucleotides or
polypeptides (e.g., enzymes) open a new field in biocatalysis. By
rapidly screening for polynucleotides encoding polypeptides of
interest, the invention provides not only a source of materials for
the development of biologics, therapeutics, and enzymes for
industrial applications, but also provides a new materials for
further processing by, for example, directed evolution and
mutagenesis to develop molecules or polypeptides modified for
particular activity, specificity or conditions.
[0047] In addition to the need for new enzymes for industrial use,
there has been a dramatic increase in the need for bioactive
compounds with novel activities. This demand has arisen largely
from changes in worldwide demographics coupled with the clear and
increasing trend in the number of pathogenic organisms that are
resistant to currently available antibiotics. For example, while
there has been a surge in demand for antibacterial drugs in
emerging nations with young populations, countries with aging
populations, such as the U.S., require a growing repertoire of
drugs against cancer, diabetes, arthritis and other debilitating
conditions. The death rate from infectious diseases has increased
58% between 1980 and 1992 and it has been estimated that the
emergence of antibiotic resistant microbes has added in excess of
$30 billion annually to the cost of health care in the U.S. alone.
(Adams et al., Chemical and Engineering News, 1995; Amann et al.,
Microbiological Reviews, 59, 1995). As a response to this trend
pharmaceutical companies have significantly increased their
screening of microbial diversity for compounds with unique
activities or specificity. Accordingly, the invention can be used
to obtain and identify polynucleotides and related sequence
specific information from, for example, infectious microorganisms
present in the environment such as, for example, in the gut of
various macroorganisms.
[0048] Identifying novel enzymes in an environmental sample is one
solution to this problem. By rapidly identifying polypeptides
having an activity of interest and polynucleotides encoding the
polypeptide of interest the invention provides methods,
compositions and sources for the development of biologics,
diagnostics, therapeutics, and compositions for industrial
applications.
[0049] The methods and compositions of the invention provide for
the identification of lead drug compounds present in an
environmental sample. The methods of the invention provide the
ability to mine the environment for novel drugs or identify related
drugs contained in different microorganisms. There are several
common sources of lead compounds (drug candidates), including
natural product collections, synthetic chemical collections, and
synthetic combinatorial chemical libraries, such as nucleotides,
peptides, or other polymeric molecules that have been identified or
developed as a result of environmental mining. Each of these
sources has advantages and disadvantages. The success of programs
to screen these candidates depends largely on the number of
compounds entering the programs, and pharmaceutical companies have
to date screened hundred of thousands of synthetic and natural
compounds in search of lead compounds. Unfortunately, the ratio of
novel to previously-discovered compounds has diminished with time.
The discovery rate of novel lead compounds has not kept pace with
demand despite the best efforts of pharmaceutical companies. There
exists a strong need for accessing new sources of potential drug
candidates. Accordingly, the invention provides a rapid and
efficient method to identify and characterize environmental samples
that contain novel drug compounds.
[0050] The majority of bioactive compounds currently in use are
derived from soil microorganisms. These compounds are generally
thought to be nonessential for growth of the organism and are
synthesized with the aid of genes involved in intermediary
metabolism hence their name--"secondary metabolites". Secondary
metabolites that influence the growth or survival of other
organisms are known as "bioactive" compounds and serve as key
components of the chemical defense arsenal of both micro- and
macro-organisms. Approximately 6,000 bioactive compounds of
microbial origin have been characterized, with more than 60%
produced by the gram positive soil bacteria of the genus
Streptomyces. (Barnes et al., Proc. Nat. Acad. Sci. U.S.A., 91,
1994). Of these, at least 70 are currently used for biomedical and
agricultural applications. The largest class of bioactive
compounds, the polyketides, include a broad range of antibiotics,
immunosuppressants and anticancer agents which together account for
sales of over $5 billion per year.
[0051] The invention provides methods of identifying a nucleic acid
sequence encoding a polypeptide having either known or unknown
function. For example, much of the diversity in microbial genomes
results from the rearrangement of gene clusters in the genome of
microorganisms. These gene clusters can be present across species
or phylogenetically related with other organisms.
[0052] 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. The gene cluster, the promoter, and additional
sequences that function in regulation altogether are referred to as
an "operon" and can include up to 20 or more genes, usually from 2
to 6 genes. Thus, a gene cluster is a group of adjacent genes that
are either identical or related, usually as to their function.
[0053] Some gene families consist of identical members. Clustering
is a prerequisite for maintaining identity between genes, although
clustered genes are not necessarily identical. Gene clusters range
from extremes where a duplication is generated to adjacent related
genes to cases where hundreds of identical genes lie in a tandem
array. Sometimes no significance is discernable in a repetition of
a particular gene. A principal example of this is the expressed
duplicate insulin genes in some species, whereas a single insulin
gene is adequate in other mammalian species.
[0054] Further, gene clusters undergo continual reorganization and,
thus, the ability to create heterogeneous libraries of gene
clusters from, for example, bacterial or other prokaryote sources
is valuable in determining sources of novel proteins, particularly
including enzymes such as, for example, the polyketide synthases
that are responsible for the synthesis of polyketides having a vast
array of useful activities. For example, polyketides are molecules
which are an extremely rich source of bioactivities, including
antibiotics (such as tetracyclines and erythromycin), anti-cancer
agents (daunomycin), immunosuppressants (FK506 and rapamycin), and
veterinary products (monensin). Many polyketides (produced by
polyketide synthases) are valuable as therapeutic agents.
Polyketide synthases are multifunctional enzymes that catalyze the
biosynthesis of a huge variety of carbon chains differing in length
and patterns of functionality and cyclization. Polyketide synthase
genes fall into gene clusters and at least one type (designated
type I) of polyketide synthases have large size genes and enzymes,
complicating genetic manipulation and in vitro studies of these
genes/proteins. Other types of proteins that are the product(s) of
gene clusters are also contemplated, including, for example,
antibiotics, antivirals, antitumor agents and regulatory proteins,
such as insulin.
[0055] The ability to select and combine desired components from a
library of polyketides and postpolyketide biosynthesis genes for
generation of novel polyketides for study is appealing. The
method(s) of the present invention make it possible to, and
facilitate the cloning of, novel polyketide synthases and other
gene clusters, since one can generate gene banks with clones
containing large inserts (especially when using the f-factor based
vectors), which facilitates cloning of gene clusters.
[0056] For example, a gene cluster can be ligated into a vector
containing an expression regulatory sequences which can control and
regulate the production of a detectable protein or protein-related
array activity from the ligated gene clusters. Use of vectors which
have an exceptionally large capacity for exogenous nucleic acid
introduction are particularly appropriate for use with such gene
clusters and are described by way of example herein to include the
f-factor (or fertility factor) of E. coli. This f-factor of E. coli
is a plasmid which affects high-frequency transfer of itself during
conjugation and is ideal to achieve and stably propagate large
nucleic acid fragments, such as gene clusters from mixed microbial
samples.
[0057] The nucleic acid isolated or derived from these samples
(e.g., a mixed population of microorganisms) or isolates thereof
can be inserted into a vector or a plasmid prior to screening of
the polynucleotides. Such vectors or plasmids are typically those
containing expression regulatory sequences, including promoters,
enhancers and the like.
[0058] Accordingly, the invention provides novel systems to clone
and screen mixed populations of organisms enriched samples, or
isolates thereof for polynucleotides encoding molecules having an
activity of interest, enzymatic activities and bioactivities of
interest in vitro. The method(s) of the invention allow the cloning
and discovery of novel bioactive molecules in vitro, and in
particular novel bioactive molecules derived from uncultivated or
cultivated samples. Large size gene clusters, genes and gene
fragments can be cloned, sequenced and screened using the method(s)
of the invention. Unlike previous strategies, the method(s) of the
invention allow one to clone screen and identify polynucleotides
and the polypeptides encoded by these polynucleotides in vitro from
a wide range of environmental samples.
[0059] The invention allows one to screen for and identify
polynucleotide sequences from complex environmental samples,
enriched samples thereof, or isolates thereof. Gene libraries can
be generated from cell free samples, so long as the sample contains
nucleic acid sequences, or from samples containing cells, cellular
material or viral particles. The organisms from which the libraries
may be prepared include prokaryotic microorganisms, such as
Eubacteria and Archaebacteria, lower eukaryotic microorganisms such
as fungi, algae and protozoa, as well as mixed populations of
plants, plant spores and pollen. The organisms may be cultured
organisms or uncultured organisms obtained from environmental
samples and includes extremophiles, such as thermophiles,
hyperthermophiles, psychrophiles and psychrotrophs.
[0060] Sources of nucleic acids used to generate a DNA library can
be obtained from environmental samples, such as, but not limited
to, microbial samples obtained from Arctic and Antarctic ice, water
or permafrost sources, materials of volcanic origin, materials from
soil or plant sources in tropical areas, droppings from various
organisms including mammals and invertebrates, as well as dead and
decaying matter and the like. The nucleic acids used to generate
the gene libraries can be obtained, for example, from enriched
subpopulations or ioslates of the sample. In another embodiment,
DNA of a plurality of isolates can be pooled to create a source of
nucleic acids for generation of the library. Alternatively, the
nucleic acids can be obtained from a plurality of isolates, a
plurality of gene libraries generated from the plurality of
isolates to obtain a plurality of gene libraries. Two or more of
the gene libraries can be pooled or combined to obtain a pooled
isolate library. Thus, for example, nucleic acids may be recovered
from either a cultured or non-cultured organism and used to produce
an appropriate gene library (e.g., a recombinant expression
library) for subsequent determination of the identity of the
particular biomolecule of interest (e.g., a polynucleotide
sequence) or screened for a bioactivity of interest (e.g., an
enzyme or biological activity).
[0061] The following outlines a general procedure for producing
libraries from both culturable and non-culturable organisms,
enriched populations, as well as mixed population of organisms and
isolates thereof, which libraries can be probed, sequenced or
screened to select therefrom nucleic acid sequences having an
identified, desired or predicted biological activity (e.g., an
enzymatic activity), which selected nucleic acid sequences can be
further evolved, mutagenized or derived.
[0062] As used herein an environmental sample is any sample
containing organisms or polynucleotides or a combination thereof.
Thus, an environmental sample can be obtained from any number of
sources (as described above), including, for example, insect feces,
hot springs, soil and the like. Any source of nucleic acids in
purified or non-purified form can be utilized as starting material.
Thus, the nucleic acids may be obtained from any source which is
contaminated by an organism or from any sample containing cells.
The environmental sample can be an extract from any bodily sample
such as blood, urine, spinal fluid, tissue, vaginal swab, stool,
amniotic fluid or buccal mouthwash from any mammalian organism. For
non-mammalian (e.g., invertebrates) organisms the sample can be a
tissue sample, salivary sample, fecal material or material in the
digestive tract of the organism. An environmental sample also
includes samples obtained from extreme environments including, for
example, hot sulfur pools, volcanic vents, and frozen tundra. The
sample can come from a variety of sources. For example, in
horticulture and agricultural testing the sample can be a plant,
fertilizer, soil, liquid or other horticultural or agricultural
product; in food testing the sample can be fresh food or processed
food (for example infant formula, seafood, fresh produce and
packaged food); and in environmental testing the sample can be
liquid, soil, sewage treatment, sludge and any other sample in the
environment which is considered or suspected of containing an
organism or polynucleotides.
[0063] When the sample is a mixture of material containing a mixed
population of organisms, for example, blood, soil or sludge, it can
be treated with an appropriate reagent which is effective to open
the cells and expose or separate the strands of nucleic acids.
Although not necessary, this lysing and nucleic acid denaturing
step will allow cloning, amplification or sequencing to occur more
readily. Further, if desired, the mixed population can be cultured
prior to analysis in order to purify or enrich a particular
population or a desired isolate (e.g., an isolate of a particular
species, genus, or family of organisms) and thus obtaining a purer
sample. This is not necessary, however. For example, culturing of
organisms in the sample can include culturing the organisms in
microdroplets and separating the cultured microdroplets with a cell
sorter into individual wells of a multi-well tissue culture plate.
Alternatively, the sample can be cultured on any number of
selective media compositions designed to inhibit or promote growth
of a particular subpopulation of organisms.
[0064] Where isolates are derived from the sample containing mixed
population of organisms, nucleic acids can be obtained from the
isolates as described below. The nucleic acids obtained from the
isolates can be used to generate a gene library or, alternatively,
be pooled with other isolate fractions of the sample wherein the
pooled nucleic acids are used to generate a gene library. The
isolates can be cultured prior to extraction of nucleic acids or
can be uncultured. Methods of isolating specific populations of
organisms present in a mixed population
[0065] Accordingly, the sample comprises nucleic acids from, for
example, a diverse and mixed population of organisms (e.g.,
microorganisms present in the gut of an insect). Nucleic acids are
isolated from the sample using any number of methods for DNA and
RNA isolation. Such nucleic acid isolation methods are commonly
performed in the art. Where the nucleic acid is RNA, the RNA can be
reversed transcribed to DNA using primers known in the art. Where
the DNA is genomic DNA, the DNA can be sheared using, for example,
a 25 gauge needle.
[0066] The nucleic acids can be cloned into an appropriate vector.
The vector used will depend upon whether the DNA is to be
expressed, amplified, sequenced or manipulated in any number of
ways known in the art (see, for example, U.S. Pat. No. 6,022,716
which discloses high throughput sequencing vectors). Cloning
techniques are known in the art or can be developed by one skilled
in the art, without undue experimentation. The choice of a vector
will also depend on the size of the polynucleotide sequence and the
host cell to be employed in the methods of the invention. Thus, the
vector used in the invention may be plasmids, phages, cosmids,
phagemids, viruses (e.g., retroviruses, parainfluenzavirus,
herpesviruses, reoviruses, paramyxoviruses, and the like), or
selected portions thereof (e.g., coat protein, spike glycoprotein,
capsid protein). For example, cosmids and phagemids are typically
used where the specific nucleic acid sequence to be analyzed or
modified is large because these vectors are able to stably
propagate large polynucleotides.
[0067] The vector containing the cloned nucleic acid sequence can
then be amplified by plating (i.e., clonal amplification) or
transfecting a suitable host cell with the vector (e.g., a phage on
an E. coli host). The cloned nucleic acid sequence is used to
prepare a library for screening (e.g., expression screening, PCR
screening, hybridization screening or the like) by transforming a
suitable organism. Hosts, known in the art are transformed by
artificial introduction of the vectors containing the nucleic acid
sequence by inoculation under conditions conducive for such
transformation. One could transform with double stranded circular
or linear nucleic acid or there may also be instances where one
would transform with single stranded circular or linear nucleic
acid sequences. By transform or transformation is meant a permanent
or transient genetic change induced in a cell following
incorporation of new DNA (e.g., DNA exogenous to the cell). Where
the cell is a mammalian cell, a permanent genetic change is
generally achieved by introduction of the DNA into the genome of
the cell. A transformed cell or host cell generally refers to a
cell (e.g., prokaryotic or eukaryotic) into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule not normally present in the host
organism.
[0068] A particularly type of vector for use in the invention
contains an f-factor origin replication. The f-factor (or fertility
factor) in E. coli is a plasmid which effects high frequency
transfer of itself during conjugation and less frequent transfer of
the bacterial chromosome itself. In a particular embodiment cloning
vectors referred to as "fosmids" or bacterial artificial chromosome
(BAC) vectors are used. These are derived from E. coli f-factor
which is able to stably integrate large segments of DNA. When
integrated with DNA from a mixed uncultured environmental sample,
this makes it possible to achieve large genomic fragments in the
form of a stable environmental gene library.
[0069] The nucleic acids derived from a mixed population or sample
may be inserted into the vector by a variety of procedures. In
general, the nucleic acid sequence is inserted into an appropriate
restriction endonuclease site(s) by procedures known in the art.
Such procedures and others are deemed to be within the scope of
those skilled in the art. A typical cloning scenario may have DNA
"blunted" with an appropriate nuclease (e.g., Mung Bean Nuclease),
methylated with, for example, EcoR I Methylase and ligated to EcoR
I linkers GGAATTCC (SEQ ID NO:1). The linkers are then digested
with an EcoR I Restriction Endonuclease and the DNA size
fractionated (e.g., using a sucrose gradient). The resulting size
fractionated DNA is then ligated into a suitable vector for
sequencing, screening or expression (e.g., a lambda vector and
packaged using an in vitro lambda packaging extract).
[0070] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0071] When the host is a eukaryote, methods of transfection or
transformation with DNA include calcium phosphate co-precipitates,
conventional mechanical procedures such as microinjection,
electroporation, insertion of a plasmid encased in liposomes, or
virus vectors, as well as others known in the art, may be used.
Eukaryotic cells can also be cotransfected with a second foreign
DNA molecule encoding a selectable marker, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (Eukaryotic Viral Vectors, Cold
Spring Harbor Laboratory, Gluzman ed., 1982). The eukaryotic cell
may be a yeast cell (e.g., Saccharomyces cerevisiae), an insect
cell (e.g., Drosophila sp.) or may be a mammalian cell, including a
human cell.
[0072] Eukaryotic systems, and mammalian expression systems, allow
for post-translational modifications of expressed mammalian
proteins to occur. Eukaryotic cells which possess the cellular
machinery for processing of the primary transcript, glycosylation,
phosphorylation, or secretion of the gene product should be used.
Such host cell lines may include, but are not limited to, CHO,
VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.
[0073] In one embodiment, once a library of clones is created using
any number of methods, including those describe above, the clones
are resuspended in a liquid media, for example, a nutrient rich
broth or other growth media known in the art. Typically the media
is a liquid media which can be readily pipetted. One or more media
types containing at least one clone of the library is then
introduced either individually or together as a mixture, into
capillaries (all or a portion thereof) in a capillary array.
[0074] In another embodiment, the library is first biopanned prior
to introduction or delivery into a capillary device or other
screening techniques. Such biopanning methods enrich the library
for sequences or activities of interest. Examples of methods for
biopanning or enrichment are described below.
[0075] In one embodiment, the library can be screened or sorted to
enrich for clones containing a sequence or activity of interested
based on polynucleotide sequences present in the library or clone.
Thus, the invention provides methods and compositions useful in
screening organisms for a desired biological activity or biological
sequence and to assist in obtaining sequences of interest that can
further be used in directed evolution, molecular biology,
biotechnology and industrial applications.
[0076] Accordingly, the invention provides methods to rapidly
screen, enrich and/or identify sequences in a sample by screening
and identifying the nucleic acid sequences present in the sample.
Thus, the invention increases the repertoire of available sequences
that can be used for the development of diagnostics, therapeutics
or molecules for industrial applications. Accordingly, the methods
of the invention can identify novel nucleic acid sequences encoding
proteins or polypeptides having a desired biological activity.
[0077] After the gene libraries (e.g., an expression library) have
been generated one can include the additional step of "biopanning"
such libraries prior to expression screening. The "biopanning"
procedure refers to a process for identifying clones having a
specified biological activity by screening for sequence homology in
a library of clones.
[0078] The probe sequence used for selectively interacting with the
target sequence of interest in the library can be a full-length
coding region sequence or a partial coding region sequence for a
known bioactivity. The library can be probed using mixtures of
probes comprising at least a portion of the sequence encoding a
known bioactivity or having a desired bioactivity. These probes or
probe libraries are preferably single-stranded. In one embodiment,
the library is preferably been converted into single-stranded form.
The probes that are particularly suitable are those derived from
DNA encoding bioactivities having an activity similar or identical
to the specified bioactivity which is to be screened. The probes
can be used to PCR amplify and thus select target sequences.
Alternatively, the probe sequences can be used as hybridization
probes which can be used to identify sequences with substantial or
a desired homology.
[0079] In another embodiment, in vivo biopanning may be performed
utilizing a FACS-based machine gene libraries or expression
libraries are constructed with vectors which contain elements which
stabilize transcribed RNA. For example, the inclusion of sequences
which result in secondary structures such as hairpins which are
designed to flank the transcribed regions of the RNA would serve to
enhance their stability, thus increasing their half life within the
cell. The probe molecules used in the biopanning process consist of
oligonucleotides labeled with detectable molecules that provide a
detectable signal upon interaction with a target sequence (e.g.,
only fluoresce upon binding of the probe to a target molecule).
Various dyes or stains well known in the art, for example those
described in "Practical Flow Cytometry", 1995 Wiley-Liss, Inc.,
Howard M. Shapiro, M. D., can be used to intercalate or associate
with nucleic acid in order to "label" the oligonucleotides. These
probes are introduced into the recombinant cells of the library
using one of several transformation methods. The probe molecules
interact or hybridize to the transcribed target mRNA or DNA
resulting in DNA/RNA heteroduplex molecules or DNA/DNA duplex
molecules. Binding of the probe to a target will yield a detectable
signal (e.g., a fluorescent signal) which is detected and sorted by
a FACS machine, or the like, during the screening process.
[0080] The probe DNA should be at least about 10 bases and
preferably at least 15 bases. In one embodiment, an entire coding
region of one part of a pathway may be employed as a probe. Where
the probe is hybridized to the target DNA in an in vitro system,
conditions for the hybridization in which target DNA is selectively
isolated by the use of at least one DNA probe will be designed to
provide a hybridization stringency of at least about 50% sequence
identity, more particularly a stringency providing for a sequence
identity of at least about 70%.
[0081] Hybridization techniques for probing a microbial DNA library
to isolate target DNA of potential interest are well known in the
art and any of those which are described in the literature are
suitable for use herein including, for example, chip-based assays,
membrane-based assays, and the like.
[0082] The resultant libraries of transformed clones can then be
further screened for clones which display an activity of interest.
Clones can be shuttled in alternative hosts for expression of
active compounds, or screened using methods described herein.
[0083] An alternative to the in vivo biopanning described above is
an encapsulation techniques such as, for example, gel
microdroplets, which may be employed to localize multiple clones in
one location to be screened on a FACS machine. Clones can then be
broken out into individual clones to be screened again on a FACS
machine to identify positive individual clones. Screening in this
manner using a FACS machine is fully described in patent
application Ser. No. 08/876,276 filed Jun. 16, 1997. Thus, for
example, if a clone mixture has a desirable activity, then the
individual clones may be recovered and rescreened utilizing a FACS
machine to determine which of such clones has the specified
desirable activity.
[0084] Different types of encapsulation strategies and compounds or
polymers can be used with the present invention. For instance, high
temperature agarose can be employed for making microdroplets stable
at high temperatures, allowing stable encapsulation of cells
subsequent to heat-kill steps utilized to remove all background
activities when screening for thermostable bioactivities.
Encapsulation can be in beads, high temperature agaroses, gel
microdroplets, cells, such as ghost red blood cells or macrophages,
liposomes, or any other means of encapsulating and localizing
molecules.
[0085] For example, methods of preparing liposomes have been
described (e.g., U.S. Pat. Nos. 5,653,996, 5,393,530 and
5,651,981), as well as the use of liposomes to encapsulate a
variety of molecules (e.g., U.S. Pat. Nos. 5,595,756, 5,605,703,
5,627,159, 5,652,225, 5,567,433, 4,235,871, 5,227,170). Entrapment
of proteins, viruses, bacteria and DNA in erythrocytes during
endocytosis has been described, as well (see, for example, Journal
of Applied Biochemistry 4, 418-435 (1982)). Erythrocytes employed
as carriers in vitro or in vivo for substances entrapped during
hypo-osmotic lysis or dielectric breakdown of the membrane have
also been described (reviewed in Ihler, G. M. (1983) J. Pharm.
Ther). These techniques are useful in the present invention to
encapsulate samples in a microenvironment for screening.
[0086] "Microenvironment," as used herein, is any molecular
structure which provides an appropriate environment for
facilitating the interactions necessary for the method of the
invention. An environment suitable for facilitating molecular
interactions include, for example, liposomes. Liposomes can be
prepared from a variety of lipids including phospholipids,
glycolipids, steroids, long-chain alkyl esters; e.g., alkyl
phosphates, fatty acid esters; e.g., lecithin, fatty amines and the
like. A mixture of fatty material may be employed such a
combination of neutral steroid, a charge amphiphile and a
phospholipid. Illustrative examples of phospholipids include
lecithin, sphingomyelin and dipalmitoylphos-phatidylcholine.
Representative steroids include cholesterol, cholestanol and
lanosterol. Representative charged amphiphilic compounds generally
contain from 12-30 carbon atoms. Mono- or dialkyl phosphate esters,
or alkyl amines; e.g., dicetyl phosphate, stearyl amine, hexadecyl
amine, dilauryl phosphate, and the like.
[0087] Further, it is possible to combine some or all of the above
embodiments such that a normalization step is performed prior to
generation of the expression library, the expression library is
then generated, the expression library so generated is then
biopanned, and the biopanned expression library is then screened
using a high throughput cell sorting and screening instrument. Thus
there are a variety of options, including: (i) generating the
library and then screen it; (ii) normalize the target DNA, generate
the library and screen it; (iii) normalize, generate the library,
biopan and screen; or (iv) generate, biopan and screen the library.
The nucleic acids used to generate a library can be obtained, for
example, from environmental samples, mixed populations of organisms
(e.g., cultured or uncultured), enriched populations thereof, and
isolates thereof. In addition, the screening techniques include,
for example, hybridization screening, PCR screening, expression
screening, and the like.
[0088] The gel microdroplet technology has had significance in
amplifying the signals available in flow cytometric analysis, and
in permitting the screening of microbial strains in strain
improvement programs for biotechnology. Wittrup et al.,
(Biotechnolo.Bioeng. (1993) 42:351-356) developed a
microencapsulation selection method which allows the rapid and
quantitative screening of >10.sup.6 yeast cells for enhanced
secretion of Aspergillus awamori glucoamylase. The method provides
a 400-fold single-pass enrichment for high-secretion mutants.
[0089] Gel microdroplet or other related technologies can be used
in the present invention to localize, sort as well as amplify
signals in the high throughput screening of recombinant libraries.
Cell viability during the screening is not an issue or concern
since nucleic acid can be recovered from the microdroplet.
[0090] Following any number of biopanning techniques capable of
enriching the library population for clones containing sequences of
interest, the enriched clones are suspended in a liquid media such
as a nutrient broth or other growth media. Accordingly, the
enriched clones comprise a plurality of host cells transformed with
constructs comprising vectors into which have been incorporated
nucleic acid sequences derived from a sample (e.g., mixed
poulations of organisms, isolates thereof, and the like). Liquid
media containing a subset of clones and one or more substrates
having a detectable molecule (e.g., an enzyme substrate) is then
introduced or contacted, either individually or together as a
mixture, with the enriched clones (e.g., into capillaries in a
capillary array). Interaction (including reaction) of the substrate
and a clone expressing an enzyme having the desire enzyme activity
produces a product or a detectable signal, which can be spatially
detected to identify one or more clones or capillaries containing
at least one signal-producing clone. The signal-producing clones or
nucleic acids contained in the signal-producing clone can then be
recovered using any number of techniques.
[0091] A "substrate" as used herein includes, for example,
substrates for the detection of a bioactivity or biomolecule (e.g.,
an enzymes and their specific enzyme activities). Such substrates
are well known in the art. For example, various enzymes and
suitable substrates specific for such enzymes are provided in
Molecular Probes, Handbook Of Fluorescent Probes and Research
Chemical (Molecular Probes, Inc.; Eugene, Oreg.), the disclosure of
which is incorporated herein by reference. The substrate can have a
detectable molecule associated with it including, for example,
chromagenic or fluorogenic molecules. A suitable substrate for use
in the present invention is any substrate that produces an
optically detectable signal upon interaction (e.g., reaction) with
a given enzyme having a desired activity, or a given clone encoding
such enzyme.
[0092] One skilled in the art can choose a suitable substrate based
on a desired enzyme activity, for example. Examples of desired
enzymes/enzymatic activities include those listed herein. A desired
enzyme activity may also comprise a group of enzymes in an
enzymatic pathway for which there exists an optical signal
substrate. One example of this is the set of carotenoid synthesis
enzymes.
[0093] Substrates are known and/or are commercially available for
glycosidases, proteases, phosphtases, and monoxygenases, among
others. Among the proteases with detectable (e.g., optical) signal
substrates are the serine proteases--trypsin and chymotrypsin.
Among the glucosidases are mannosidase, amyloglucosidase,
cellulase, neuraminidase, beta-galactosidase, beta-glucosidase,
beta-glucouronidase and alpha-amylase.
[0094] Where the desired activity is in the same class as that of
other biomolecules or enzymes having a number known substrates, the
activity can be examined using a cocktail of the known substrates.
For example, substrates are known for approximately 20 commercially
available esterases and the combination of these known substrate
can provide detectable, if not optimal, signal production.
[0095] The optical signal substrate can be a chromogenic substrate,
a fluorogenic substrate, a bio-or chemi-luminescent substrate, or a
fluorescence resonance energy transfer (FRET) substrate. The
detectable species can be one which results from cleavage of the
substrate or a secondary molecule which is so affected by the
cleavage or other substrate/biomolecule interaction as to undergo a
detectable change. Innumerable examples of detectable assay formats
are known from the diagnostic arts which use immmunoassay,
chromogenic assay, and labeled probe methodologies.
[0096] In one embodiment, the optical signal substrate can be a
bio- or chemi-luminescent substrate. Chemiluminescent substrates
for several enzymes are available from Tropix (Bedford, Mass.).
Among the enzymes having known chemiluminescent substrates are
alkaline phosphatase, beta-galactosidase, beta-glucouronidase, and
beta-glucosidase.
[0097] In another embodiment, chromogenic substrates may be used,
particularly for certain enzymes such as hydrolytic enzymes. For
example, the optical signal substrate can be an indolyl derivative,
which is enyzmatically cleaved to yield a chromogenic product.
Where chromogenic substrate are used, the optically detectable
signal is optical absorbance (including changes in absorbance). In
this embodiment, signal detection can be provided by an absorbance
measurement using a spectrophotometer or the like.
[0098] In another embodiment, a fluorogenic substrate is used, such
that the optically detectable signal is fluorescence. Fluorogenic
substrates provide high sensitivity for improved detection, as well
as alternate detection modes. Hydroxy- and amino-substituted
coumarins are the most widely used fluorophores used for preparing
fluorogenic substrates. A typical coumarin-based fluorogenic
substrate is 7-hydroxycoumarin, commonly known as umbelliferone
(Umb). Derivatives and analogs of umbelliferone are also used.
Substrate based on derivative and analogs of florescein (such as
FDG or C12-FDG) and rhodamine are also used. Substrates derived
from resorufin (e.g., resorufin beta-D-galactopyranoside or
resorufin beta-D-glucouronide) are particularly useful in the
present invention. Resorufin-based substrates are useful, for
example, in screening for glycosidases, hydrolases and dealkylases.
Lipophilic derivatives of the foregoing substrates (e.g., alkylated
derivatives) may be useful in certain embodiments, since they
generally load more readily into cells and may tend to associate
with lipid regions of the cell. Fluorescein and resorufin are
available commercially as alkylated derivatives that form products
that are relatively insoluble in water (i.e., lipophilic). For
example, fluorescence imaging can be performed using C12-resorufin
galactoside, produced by Molecular Probes (Eugene, Oreg.) as a
substrate.
[0099] The particular fluorogenic substrate used may be chosen
based on the enzymatic activity being screened. For examples:
[0100] Lipases/esterases. When screening for an enzyme having
lipase or esterase activity, an acylated derivatives of fluorescein
in used. The fluorophore is hydrolyzed from the derivative to
generate a signal.
[0101] Proteases. Enzymes having protease activity can be screened
in the same way as the esterases, with an amide bond cleaved
instead of an ester. There are now well over 100 different protease
substrates available with an acylated fluorophore at the scissile
bond. Rhodamine derivatives are generally used.
[0102] Monooxygenases (dealkylases). Several coumarin derivatives
suitable as monooxygenase substrates are commercially available.
Typically, in these substrates, the hydroxylation of the ethyl
group in the compound results in the release of the resorufin
fluorophore.
[0103] Typically, the substrates are able to enter the cell and
maintain its presence within the cell for a period sufficient for
analysis to occur (e.g., once the substrate is in the cell it does
not "leak" back out before reacting with the enzyme being screened
to an extend sufficient to produce a detectable response).
Retention of the substrate in the cell can be enhanced by a variety
of techniques. In one method, the substrate compound is
structurally modified by addition of a hydrophobic (e.g., alkyl)
tail. In another embodiment, a solvent, such as DMSO or glycerol,
can be used to coat the exterior of the cell. Also the substrate
can be administered to the cells at reduced temperature, which has
been observed to retard leakage of substrates from cells. However,
entry of the substrate into the cell is not necessary where, for
example, the enzyme or polypeptide is secreted, present in a lysed
cellular sample or the like, or where the substrate can act
externally to the cell (e.g., an extracellular receptor-ligand
complex).
[0104] The optical signal substrate can, in some embodiments, be a
FRET substrate. FRET is a spectroscopic method that can monitor
proximity and relative angular orientation of fluorophores. A
fluorescent indicator system that uses FRET to measure the
concentration of a substrate or products includes two fluorescent
moieties having emission and excitation spectra that render one a
"donor" fluorescent moiety and the other an "acceptor" fluorescent
moiety. The two fluorescent moieties are chosen such that the
excitation spectrum of the acceptor fluorescent moiety overlaps
with the emission spectrum of the excited moiety (the donor
fluorescence moiety). The donor moiety is excited by light of
appropriate intensity within the excitation spectrum of the donor
moiety and emits the absorbed energy as fluorescent light. When the
acceptor fluorescent protein moiety is positioned to quench the
donor moiety in the excited state, the fluorescence energy is
transferred to the acceptor moiety, which can emit a second photon.
The emission spectra of the donor and acceptor moieties have
minimal overlap so that the two emissions can be distinguished.
Thus, when acceptor emits fluorescence at longer wavelength that
the donor, then the net steady state effect is that the donor's
emission is quenched, and the acceptor now emits when excited at
the donor's absorption maximum.
[0105] The detectable or optical signal can be measured using, for
example, a fluoremeter (or the like) to detect fluorescence,
including fluorescence polarization, time-resolved fluorescence or
FRET. In general, excitation radiation, from an excitation source
having a first wavelength, causes the excitation radiation to
excite the sample. In response, fluorescence compounds in the
sample emit radiation having a wavelength that is different from
the excitation wavelength. Methods of performing assays on
fluorescent materials are well known in the art and are described,
e.g., by Lakowicz (Principles of Fluorescence Spectroscopy, New
York, Plenum Press, 1983) and Herman ("Resonance energy transfer
microscopy," in: Fluorescence Microscopy of Living Cells in
Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor &
Wang, San Diego, Academic Press, 1989, pp. 219-243). Examples of
fluorescence detection techniques are described in further detail
below.
[0106] In addition, several methods have been described in the
literature for using reporter genes to measure gene expression.
Nolan et al. describes a technique to analyze beta-galactosidase
expression in mammalian cells. This technique employs
fluorescein-di-beta-D-glactopyran- oside (FDG) as a substrate for
beta-galactosidase, which releases fluorescein, a product that can
be detected by its fluorescence emission upon hydrolysis (Nolan et
al., 1991). Other fluorogenic substrates have been developed, such
as 5-dodecanoylamino fluorescein di-beta-Dgalactopyranside
(C12-FDG) (Molecular Probes), which differs from FDG in that it is
a lipophilic fluorescein derivative that can easily cross most cell
membranes under physiological culture conditions.
[0107] The above-mentioned beta-galactosidase assays may be
employed to screen single E. coli cells, expressing recombinant
beta-D-galactosidase isolated, for example, from a
hyperthermophilic archaeon such as Sulfolobus solfataricus. Other
reporter genes may be useful as substrates and are known for
beta-glucouronidase, alkaline phosphatase, chloramphenical
acetyltransferase (CAT) and luciferase.
[0108] The library may, for example, be screened for a specified
enzyme activity. For example, the enzyme activity screened for may
be one or more of the six IUB classes; oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases. The
recombinant enzymes which are determined to be positive for one or
more of the IUB classes may then be rescreened for a more specific
enzyme activity.
[0109] Alternatively, the library may be screened for a more
specialized enzyme activity. For example, instead of generically
screening for hydrolase activity, the library may be screened for a
more specialized activity, i.e. the type of bond on which the
hydrolase acts. Thus, for example, the library may be screened to
ascertain those hydrolases which act on one or more specified
chemical functionalities, such as: (a) amide (peptide bonds), i.e.
proteases; (b) ester bonds, i.e. esterases and lipases; (c)
acetals, i.e., glycosidases and the like.
[0110] As described with respect to one of the above aspects, the
invention provides a process for activity screening of clones
containing selected DNA derived from a microorganism which method
includes:
[0111] screening a library for a biomolecule of interest or
bioactivity of interest, wherein the library includes a plurality
of clones, the clones having been prepared by recovering nucleic
acids (e.g., genomic DNA) from a mixed population of organisms,
enriched populations thereof, or isolates thereof, and transforming
a host with the nucleic acids to produce clones which are screened
for the biomolecule or bioactivity of interest.
[0112] In another embodiment, an enrichment step may be used before
activity based screening. The enrichment step can be, for example,
a biopanning method. This procedure of "biopanning" is described
and exemplified in U.S. Pat. No. 6,054,002, issued Apr. 25, 2000,
which is incorporated herein by reference.
[0113] In another embodiment, polynucleotides are contained in
clones, the clones having been prepared from nucleic acid sequences
of a mixed population of organisms, wherein the nucleic acid
sequences are used to prepare a gene library of the mixed
population of organisms. The gene library is screened for a
sequence of interest by transfecting a host cell containing the
library with at least one nucleic acid sequence having a detectable
molecule which is all or a portion of a DNA sequence encoding a
bioactivity having a desirable activity and separating the library
clones containing the desirable sequence by, for example, a
fluorescent based analysis.
[0114] The present invention offers the ability to screen for many
types of bioactivities. For instance, the ability to select and
combine desired components from a library of polyketides and
postpolyketide biosynthesis genes for generation of novel
polyketides for study is appealing. The method(s) of the present
invention make it possible to and facilitate the cloning of novel
polyketide synthases, and other relevant pathways or genes encoding
commercially relevant secondary metabolites, since one can generate
gene libraries with clones containing large inserts (especially
when using vectors which can accept large inserts, such as the
f-factor based vectors), which facilitates cloning of gene
clusters.
[0115] The biopanning approach described above can be used to
create libraries enriched with clones carrying sequences homologous
to a given probe sequence. Using this approach libraries containing
clones with inserts of up to 40 kbp can be enriched approximately
1,000 fold after each round of panning. This enables one to reduce
the number of clones to be screened after I round of biopanning
enrichment. This approach can be applied to create libraries
enriched for clones carrying sequence of interest related to a
bioactivity of interest for example polyketide sequences.
[0116] Hybridization screening using high density filters or
biopanning has proven an efficient approach to detect homologues of
pathways containing conserved genes. To discover novel bioactive
molecules that may have no known counterparts, however, other
approaches are necessary. Another approach of the present invention
is to screen in E. coli for the expression of small molecule ring
structures or "backbones". Because the genes encoding these
polycyclic structures can often be expressed in E. coli the small
molecule backbone can be manufactured albeit in an inactive form.
Bioactivity is conferred upon transferring the molecule or pathway
to an appropriate host that expresses the requisite glycosylation
and methylation genes that can modify or "decorate" the structure
to its active form. Thus, inactive ring compounds, recombinantly
expressed in E. Coli are detected to identify clones which are then
shuttled to a metabolically rich host, such as Streptomyces, for
subsequent production of the bioactive molecule. The use of high
throughput robotic systems allows the screening of hundreds of
thousands of clones in multiplexed arrays in microliter dishes.
[0117] One approach to detect and enrich for clones carrying these
structures is to use the capillary screening methods or FACS
screening, a procedure described and exemplified in U.S. Ser. No.
08/876,276, filed Jun. 16, 1997. Polycyclic ring compounds
typically have characteristic fluorescent spectra when excited by
ultraviolet light. Thus, clones expressing these structures can be
distinguished from background using a sufficiently sensitive
detection method. For example, high throughput FACS screening can
be utilized to screen for small molecule backbones in E. coli
libraries. Commercially available FACS machines are capable of
screening up to 100,000 clones per second for UV active molecules.
These clones can be sorted for further FACS screening or the
resident plasmids can be extracted and shuttled to Streptomyces for
activity screening.
[0118] In an alternate screening approach, after shuttling to
Streptomyces hosts, organic extracts from candidate clones can be
tested for bioactivity by susceptibility screening against test
organisms such as Staphylococcus aureus, E. coli, or Saccharomyces
cervisiae. FACS screening can be used in this approach by
co-encapsulating clones with the test organism.
[0119] An alternative to the above-mentioned screening methods
provided by the present invention is an approach termed "mixed
extract" screening. The "mixed extract" screening approach takes
advantage of the fact that the accessory genes needed to confer
activity upon the polycyclic backbones are expressed in
metabolically rich hosts, such as Streptomyces, and that the
enzymes can be extracted and combined with the backbones extracted
from E. coli clones to produce the bioactive compound in vitro.
Enzyme extract preparations from metabolically rich hosts, such as
Streptomyces strains, at various growth stages are combined with
pools of organic extracts from E. coli libraries and then evaluated
for bioactivity.
[0120] Another approach to detect activity in the E. coli clones is
to screen for genes that can convert bioactive compounds to
different forms. For example, a recombinant enzyme was recently
discovered that can convert the low value daunomycin to the higher
value doxorubicin. Similar enzyme pathways are being sought to
convert penicillins to cephalosporins.
[0121] Capillary screening, for example, can also be used to detect
expression of UV fluorescent molecules in metabolically rich hosts,
such as Streptomyces. Recombinant oxytetracylin retains its
diagnostic red fluorescence when produced heterologously in S.
lividans TK24. Pathway clones, which can be identified by the
methods and systems of the invention, can thus be screened for
polycyclic molecules in a high throughput fashion.
[0122] Recombinant bioactive compounds can also be screened in vivo
using "two-hybrid" systems, which can detect enhancers and
inhibitors of protein-protein or other interactions such as those
between transcription factors and their activators, or receptors
and their cognate targets. In this embodiment, both a small
molecule pathway and a GFP reporter construct are co-expressed.
Clones altered in GFP expression can then be identified and the
clone isolated for characterization.
[0123] As indicated, common approaches to drug discovery involve
screening assays in which disease targets (macromolecules
implicated in causing a disease) are exposed to potential drug
candidates which are tested for therapeutic activity. In other
approaches, whole cells or organisms that are representative of the
causative agent of the disease, such as bacteria or tumor cell
lines, are exposed to the potential candidates for screening
purposes. Any of these approaches can be employed with the present
invention.
[0124] The present invention also allows for the transfer of cloned
pathways derived from uncultivated samples into metabolically rich
hosts for heterologous expression and downstream screening for
bioactive compounds of interest using a variety of screening
approaches briefly described above.
[0125] After viable or non-viable cells, each containing a
different expression clone from the gene library are screened, and
positive clones are recovered, DNA can be isolated from positive
clones utilizing techniques well known in the art. The DNA can then
be amplified either in vivo or in vitro by utilizing any of the
various amplification techniques known in the art. In vivo
amplification would include transformation of the clone(s) or
subclone(s) into a viable host, followed by growth of the host. In
vitro amplification can be performed using techniques such as the
polymerase chain reaction. Once amplified the identified sequences
can be "evolved" or sequenced.
[0126] One advantage afforded by present invention is the ability
to manipulate the identified biomolecules or bioactivities to
generate and select for encoded variants with altered sequence,
activity or specificity.
[0127] Clones found to have biomolecules or bioactivities for which
the screen was performed can be subjected to directed mutagenesis
to develop new biomolecules or bioactivities with desired
properties or to develop modified biomolecules or bioactivities
with particularly desired properties that are absent or less
pronounced in nature (e.g., wild-type activity), such as stability
to heat or organic solvents. Any of the known techniques for
directed mutagenesis are applicable to the invention. For example,
particularly preferred mutagenesis techniques for use in accordance
with the invention include those described below.
[0128] Alternatively, it may be desirable to variegate a
biomolecule (e.g., a peptide, protein, or polynucleotide sequence)
or a bioactivity (e.g., an enzymatic activity) obtained, identified
or cloned as described herein. Such variegation can modify the
biomolecule or bioactivity in order to increase or decrease, for
example, a polypeptide's activity, specificity, affinity, function,
and the like. DNA shuffling can be used to increase variegation in
a particular sample. DNA shuffling is meant to indicate
recombination between substantially homologous but non-identical
sequences, in some embodiments DNA shuffling may involve crossover
via non-homologous recombination, such as via cer/lox and/or
flp/frt systems and the like (see, for example, U.S. Pat. No.
5,939,250, issued to Dr. Jay Short on Aug. 17, 1999, and assigned
to Diversa Corporation, the disclosure of which is incorporated
herein by reference). Various methods for shuffling, mutating or
variegating polynucleotide or polypeptide sequences are discussed
below.
[0129] Nucleic acid shuffling is a method for in vitro or in vivo
homologous recombination of pools of shorter or smaller
polynucleotides to produce a polynucleotide or polynucleotides.
Mixtures of related nucleic acid sequences or polynucleotides are
subjected to sexual PCR to provide random polynucleotides, and
reassembled to yield a library or mixed population of recombinant
hybrid nucleic acid molecules or polynucleotides. In contrast to
cassette mutagenesis, only shuffling and error-prone PCR allow one
to mutate a pool of sequences blindly (without sequence information
other than primers).
[0130] The advantage of the mutagenic shuffling of the invention
over error-prone PCR alone for repeated selection can best be
explained as follows. Consider DNA shuffling as compared with
error-prone PCR (not sexual PCR). The initial library of selected
or pooled sequences can consist of related sequences of diverse
origin or can be derived by any type of mutagenesis (including
shuffling) of a single gene. A collection of selected sequences is
obtained after the first round of activity selection. Shuffling
allows the free combinatorial association of all of the related
sequences, for example.
[0131] 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.
[0132] Since cross-overs occur at regions of homology,
recombination will primarily occur between members of the same
sequence family. This discourages combinations of sequences that
are grossly incompatible (e.g., having different activities or
specificities). It is contemplated that multiple families of
sequences can be shuffled in the same reaction. Further, shuffling
generally conserves the relative order.
[0133] Rare shufflants will contain a large number of the best
molecules (e.g., highest activity or specificity) and these rare
shufflants may be selected based on their superior activity or
specificity.
[0134] A pool of 100 different polypeptide sequences can be
permutated in up to 10.sup.3 different ways. This large number of
permutations cannot be represented in a single library of DNA
sequences. Accordingly, it is contemplated that multiple cycles of
DNA shuffling and selection may be required depending on the length
of the sequence and the sequence diversity desired. Error-prone
PCR, in contrast, keeps all the selected sequences in the same
relative orientation, generating a much smaller mutant cloud.
[0135] The template polynucleotide which may be used in the methods
of the invention may be DNA or RNA. It may be of various lengths
depending on the size of the gene or shorter or smaller
polynucleotide to be recombined or reassembled. 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 the invention,
and in fact have been successfully used.
[0136] The template polynucleotide may be obtained by amplification
using the PCR reaction (U.S. Pat. Nos. 4,683,202 and 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.
[0137] The template polynucleotide often is double-stranded. A
double-stranded nucleic acid molecule is recommended to ensure that
regions of the resulting single-stranded polynucleotides are
complementary to each other and thus can hybridize to form a
double-stranded molecule.
[0138] 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.
[0139] 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.
[0140] Alternatively, it is also contemplated that double-stranded
nucleic acid having multiple nicks may be used in the methods of
the invention. A nick is a break in one strand of the
double-stranded nucleic acid. The distance between such nicks is
preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.
This can provide areas of self-priming to produce shorter or
smaller polynucleotides to be included with the polynucleotides
resulting from random primers, for example.
[0141] 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.
[0142] The number of different specific polynucleotides in the
mixture will be at least about 100, preferably at least about 500,
and more preferably at least about 1000.
[0143] 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.
[0144] 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.
[0145] 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 may be added in a 20 fold excess by weight as
compared to the total nucleic acid, more preferably the
single-stranded polynucleotides may be added in a 10 fold excess by
weight as compared to the total nucleic acid.
[0146] 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.
[0147] 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.
[0148] 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 and pH.
[0149] 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.
[0150] 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%.
[0151] 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.
[0152] 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.
[0153] The polymerase may be added to the random polynucleotides
prior to annealing, simultaneously with annealing or after
annealing.
[0154] 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.
[0155] 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.
[0156] This larger polynucleotides may contain a number of copies
of a polynucleotide having the same size as the template
polynucleotide in tandem. This concatemeric polynucleotide is then
denatured into single copies of the template polynucleotide. The
result will be a population of polynucleotides of approximately the
same size as the template polynucleotide. The population will be a
mixed population where single or double-stranded polynucleotides
having an area of identity and an area of heterology have been
added to the template polynucleotide prior to shuffling. These
polynucleotides are then cloned into the appropriate vector and the
ligation mixture used to transform bacteria.
[0157] 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. Nos. 4,683,195 and
4,683,202), rather than by digestion of the concatemer.
[0158] 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.
[0159] 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.
[0160] For example, if a polynucleotide, identified by the methods
of described herein, encodes a protein with a first binding
affinity, subsequent mutated (e.g., shuffled) sequences having an
increased binding efficiency to a ligand may be desired. In such a
case 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] Any source of nucleic acid, in a 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 50,000 base pairs. It is contemplated that
entire vectors containing the nucleic acid encoding the protein of
interest may be used in the methods of the invention.
[0165] Any specific nucleic acid sequence can be used to produce
the population of hybrids by the present process. It is only
necessary that a small population of hybrid sequences of the
specific nucleic acid sequence exist or be available for the
present process.
[0166] A population of specific nucleic acid sequences having
mutations may be created by a number of different methods.
Mutations may be created by error-prone PCR. Error-prone PCR uses
low-fidelity polymerization conditions to introduce a low level of
point mutations randomly over a long sequence. 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.
[0167] Alternatively, a small mixed population of specific nucleic
acids may be found in nature in that they may consist of different
alleles of the same gene or the same gene from different related
species (i.e., cognate genes). Alternatively, they may be related
DNA sequences found within one species, for example, the
immunoglobulin genes.
[0168] Once a mixed population of 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.
[0169] The choice of vector depends on the size of the
polynucleotide sequence and the host cell to be employed in the
methods of the invention. The templates of the invention may be
plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses,
parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and
the like), or selected portions thereof (e.g., coat protein, spike
glycoprotein, capsid protein). 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.
[0170] If a mixed population of the specific nucleic acid sequence
is cloned into a vector it can be clonally amplified. Utility can
be readily determined by screening expressed polypeptides.
[0171] The DNA shuffling method of the 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.
[0172] 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.
[0173] 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
(e.g., 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.
[0174] 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.
[0175] It is also contemplated that the method of the 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 typically includes sequences
of about 40 kb. Amplification of a whole genome such as that of E.
coli (5, 000 kb) by PCR would require about 250 primers yielding
125 forty kb polynucleotides. 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 concatamer
containing many copies of the genome.
[0176] A 100 fold amplification in the copy number and an average
polynucleotide size of greater than 50 kb may be obtained when only
random polynucleotides are used. It is thought that the larger
concatamer is generated by overlap of many smaller polynucleotides.
The quality of specific PCR products obtained using synthetic
primers will be indistinguishable from the product obtained from
unamplified DNA. It is expected that this approach will be useful
for the mapping of genomes.
[0177] The polynucleotide to be shuffled can be produced as random
or non-random polynucleotides, at the discretion of the
practitioner. Moreover, the invention provides a method of
shuffling that is applicable to a wide range of polynucleotide
sizes and types, including the step of generating polynucleotide
monomers to be used as building blocks in the reassembly of a
larger polynucleotide. For example, the building blocks can be
fragments of genes or they can be comprised of entire genes or gene
pathways, or any combination thereof.
[0178] In an embodiment of in vivo shuffling, a mixed population of
a specific nucleic acid sequence is introduced into bacterial or
eukaryotic cells under conditions such that at least two different
nucleic acid sequences are present in each host cell. The
polynucleotides can be introduced into the host cells by a variety
of different methods. The host cells can be transformed with the
smaller polynucleotides using methods known in the art, for example
treatment with calcium chloride. If the polynucleotides are
inserted into a phage genome, the host cell can be transfected with
the recombinant phage genome having the specific nucleic acid
sequences. Alternatively, the nucleic acid sequences can be
introduced into the host cell using electroporation, transfection,
lipofection, biolistics, conjugation, and the like.
[0179] In general, in this embodiment, specific nucleic acid
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.
[0180] 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.
[0181] 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 one
embodiment, some of the specific nucleic acid sequences are present
on linear polynucleotides.
[0182] 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.
[0183] 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. 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.
[0184] 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.
[0185] In another embodiment of the invention, it is contemplated
that during the first round a subset of specific nucleic acid
sequences can be generated as smaller polynucleotides by slowing or
halting their PCR amplification prior to introduction into the host
cell. The size of the polynucleotides must be large enough to
contain some regions of identity with the other sequences so as to
homologously recombine with the other sequences. The size of the
polynucleotides will range from 0.03 kb to 100 kb more preferably
from 0.2 kb to 10 kb. 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.
[0186] The shorter polynucleotide sequences can be single-stranded
or double-stranded. The reaction conditions suitable for separating
the strands of nucleic acid are well known in the art.
[0187] The steps of this process can be repeated indefinitely,
being limited only by the number of possible hybrids which can be
achieved.
[0188] 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 one 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.
[0189] The population of vectors containing the pool of mutated
nucleic acid sequences is introduced into the E. coli host cells.
The vector nucleic acid sequences may be introduced by
transformation, transfection or infection in the case of phage. The
concentration of vectors used to transform the bacteria is such
that a number of vectors is introduced into each cell. Once present
in the cell, the efficiency of homologous recombination is such
that homologous recombination occurs between the various vectors.
This results in the generation of hybrids (daughters) having a
combination of mutations which differ from the original parent
mutated sequences. The host cells are then clonally replicated and
selected for the marker gene present on the vector. Only those
cells having a plasmid will grow under the selection. The host
cells which contain a vector are then tested for the presence of
favorable mutations.
[0190] 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.
[0191] The parent mutated specific nucleic acid population, either
as polynucleotides or cloned into the same vector is introduced
into the host cells already containing the daughter nucleic acids.
Recombination is allowed to occur in the cells and the next
generation of recombinants, or granddaughters are selected by the
methods described above. This cycle can be repeated a number of
times until the nucleic acid or peptide having the desired
characteristics is obtained. It is contemplated that in subsequent
cycles, the population of mutated sequences which are added to the
hybrids may come from the parental hybrids or any subsequent
generation.
[0192] 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 the invention
provide a means of doing so.
[0193] 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.
[0194] 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.
[0195] In a another embodiment, the invention provides for a method
for shuffling, assembling, reassembling, recombining, and/or
concatenating at least two polynucleotides to form a progeny
polynucleotide (e.g., a chimeric progeny polynucleotide that can be
expressed to produce a polypeptide or a gene pathway). In a
particular embodiment, a double stranded polynucleotide (e.g., two
single stranded sequences hybridized to each other as hybridization
partners) is treated with an exonuclease to liberate nucleotides
from one of the two strands, leaving the remaining strand free of
its original partner so that, if desired, the remaining strand may
be used to achieve hybridization to another partner.
[0196] In a particular aspect, a double stranded polynucleotide end
(that may be part of--or connected to--a polynucleotide or a
non-polynucleotide sequence) is subjected to a source of
exonuclease activity. Enzyme with 3' exonuclease activity, an
enzyme with 5' exonuclease activity, an enzyme with both 3'
exonuclease activity and 5' exonuclease activity, and any
combination thereof can be used in the invention. An exonuclease
can be used to liberate nucleotides from one or both ends of a
linear double stranded polynucleotide, and from one to all ends of
a branched polynucleotide having more than two ends.
[0197] By contrast, a non-enzymatic step may be used to shuffle,
assemble, reassemble, recombine, and/or concatenate polynucleotide
building blocks that is comprised of subjecting a working sample to
denaturing (or "melting") conditions (for example, by changing
temperature, pH, and/or salinity conditions) so as to melt a
working set of double stranded polynucleotides into single
polynucleotide strands. For shuffling, it is desirable that the
single polynucleotide strands participate to some extent in
annealment with different hybridization partners (i.e. and not
merely revert to exclusive re-annealment between what were former
partners before the denaturation step). The presence of the former
hybridization partners in the reaction vessel, however, does not
preclude, and may sometimes even favor, re-annealment of a single
stranded polynucleotide with its former partner, to recreate an
original double stranded polynucleotide.
[0198] In contrast to this non-enzymatic shuffling step comprised
of subjecting double stranded polynucleotide building blocks to
denaturation, followed by annealment, the invention further
provides an exonuclease-based approach requiring no denaturation
rather, the avoidance of denaturing conditions and the maintenance
of double stranded polynucleotide substrates in annealed (i.e.
non-denatured) state are necessary conditions for the action of
exonucleases (e.g., exonuclease III and red alpha gene product). In
further contrast, the generation of single stranded polynucleotide
sequences capable of hybridizing to other single stranded
polynucleotide sequences is the result of covalent cleavage--and
hence sequence destruction--in one of the hybridization partners.
For example, an exonuclease III enzyme may be used to enzymatically
liberate 3' terminal nucleotides in one hybridization strand (to
achieve covalent hydrolysis in that polynucleotide strand); and
this favors hybridization of the remaining single strand to a new
partner (since its former partner was subjected to covalent
cleavage).
[0199] It is particularly appreciated that enzymes can be
discovered, optimized (e.g., engineered by directed evolution), or
both discovered and optimized specifically for the instantly
disclosed approach that have more optimal rates and/or more highly
specific activities &/or greater lack of unwanted activities.
In fact it is expected that the invention may encourage the
discovery and/or development of such designer enzymes.
[0200] Furthermore, it is appreciated that one can protect the end
of a double stranded polynucleotide or render it susceptible to a
desired enzymatic action of an exonuclease as necessary. For
example, a double stranded polynucleotide end having a 3' overhang
is not susceptible to the exonuclease action of exonuclease III.
However, it may be rendered susceptible to the exonuclease action
of exonuclease III by a variety of means; for example, it may be
blunted by treatment with a polymerase, cleaved to provide a blunt
end or a 5' overhang, joined (ligated or hybridized) to another
double stranded polynucleotide to provide a blunt end or a 5'
overhang, hybridized to a single stranded polynucleotide to provide
a blunt end or a 5' overhang, or modified by any of a variety of
means).
[0201] According to one aspect, an exonuclease may be allowed to
act on one or on both ends of a linear double stranded
polynucleotide and proceed to completion, to near completion, or to
partial completion. When the exonuclease action is allowed to go to
completion, the result will be that the length of each 5' overhang
will be extend far towards the middle region of the polynucleotide
in the direction of what might be considered a "rendezvous point"
(which may be somewhere near the polynucleotide midpoint).
Ultimately, this results in the production of single stranded
polynucleotides (that can become dissociated) that are each about
half the length of the original double stranded polynucleotide.
[0202] Thus, the exonuclease-mediated approach is useful for
shuffling, assembling and/or reassembling, recombining, and
concatenating polynucleotide building blocks. The polynucleotide
building blocks can be up to ten bases long or tens of bases long
or hundreds of bases long or thousands of bases long or tens of
thousands of bases long or hundreds of thousands of bases long or
millions of bases long or even longer.
[0203] Substrates for an exonuclease may be generated by subjecting
a double stranded polynucleotide to fragmentation. Fragmentation
may be achieved by mechanical means (e.g., shearing, sonication,
and the like), by enzymatic means (e.g., using restriction
enzymes), and by any combination thereof. Fragments of a larger
polynucleotide may also be generated by polymerase-mediated
synthesis.
[0204] Additional examples of enzymes with exonuclease activity
include red-alpha and venom phosphodiesterases. Red alpha
(red.alpha.) gene product (also referred to as lambda exonuclease)
is of bacteriophage .lambda. origin. Red alpha gene product acts
processively from 5'-phosphorylated termini to liberate
mononucleotides from duplex DNA (Takahashi & Kobayashi, 1990).
Venom phosphodiesterases (Laskowski, 1980) is capable of rapidly
opening supercoiled DNA.
[0205] In one aspect, the present invention provides a
non-stochastic method termed synthetic ligation reassembly (SLR),
that is somewhat related to stochastic shuffling, save that the
nucleic acid building blocks are not shuffled or concatenated or
chimerized randomly, but rather are assembled
non-stochastically.
[0206] The SLR method does not depend on the presence of a high
level of homology between polynucleotides to be shuffled. The
invention can be used to non-stochastically generate libraries (or
sets) of progeny molecules comprised of over 10.sup.100 different
chimeras. Conceivably, SLR can even be used to generate libraries
comprised of over 10.sup.1000 different progeny chimeras.
[0207] Thus, in one aspect, the invention provides a non-stochastic
method of producing a set of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design, which method is comprised of the steps of generating, by
design, a plurality of specific nucleic acid building blocks having
serviceable mutually compatible ligatable ends, and assembling
these nucleic acid building blocks, such that a designed overall
assembly order is achieved.
[0208] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, in one aspect,
the overall assembly order in which the nucleic acid building
blocks can be coupled is specified by the design of the ligatable
ends and, if more than one assembly step is to be used, then the
overall assembly order in which the nucleic acid building blocks
can be coupled is also specified by the sequential order of the
assembly step(s). In a one embodiment of the invention, the
annealed building pieces are treated with an enzyme, such as a
ligase (e.g., T4 DNA ligase) to achieve covalent bonding of the
building pieces.
[0209] In a another embodiment, the design of nucleic acid building
blocks is obtained upon analysis of the sequences of a set of
progenitor nucleic acid templates that serve as a basis for
producing a progeny set of finalized chimeric nucleic acid
molecules. These progenitor nucleic acid templates thus serve as a
source of sequence information that aids in the design of the
nucleic acid building blocks that are to be mutagenized, i.e.
chimerized or shuffled.
[0210] In one exemplification, the invention provides for the
chimerization of a family of related genes and their encoded family
of related products. In a particular exemplification, the encoded
products are enzymes. As a representative list of families of
enzymes which may be mutagenized in accordance with the aspects of
the present invention, there may be mentioned, the following
enzymes and their functions: Lipase/Esterase, Protease,
Glycosidase/Glycosyl, transferase, Phosphatase/Kinase,
Mono/Dioxygenase, Haloperoxidase, Lignin, peroxidase/Diarylpropane
peroxidase, Epoxide hydrolase, Nitrile hydratase/nitrilase,
Transaminase, Amidase/Acylase. These exemplifications, while
illustrating certain specific aspects of the invention, do not
portray the limitations or circumscribe the scope of the disclosed
invention.
[0211] Thus according to one aspect of the invention, the sequences
of a plurality of progenitor nucleic acid templates identified
using the methods of the invention are aligned in order to select
one or more demarcation points, which demarcation points can be
located at an area of homology. The demarcation points can be used
to delineate the boundaries of nucleic acid building blocks to be
generated. Thus, the demarcation points identified and selected in
the progenitor molecules serve as potential chimerization points in
the assembly of the progeny molecules.
[0212] Typically a demarcation point is an area of homology
(comprised of at least one homologous nucleotide base) shared by at
least two progenitor templates, but the demarcation point can be an
area of homology that is shared by at least half of the progenitor
templates, at least two thirds of the progenitor templates, at
least three fourths of the progenitor templates, and preferably at
almost all of the progenitor templates. Even more preferably still
a demarcation point is an area of homology that is shared by all of
the progenitor templates.
[0213] In another embodiment, the ligation reassembly process is
performed exhaustively in order to generate an exhaustive library.
In other words, all possible ordered combinations of the nucleic
acid building blocks are represented in the set of finalized
chimeric nucleic acid molecules. At the same time, the assembly
order (i.e. the order of assembly of each building block in the 5'
to 3 sequence of each finalized chimeric nucleic acid) in each
combination is by design (or non-stochastic). Because of the
non-stochastic nature of the invention, the possibility of unwanted
side products is greatly reduced.
[0214] In yet another embodiment, the invention provides that, the
ligation reassembly process is performed systematically, for
example in order to generate a systematically compartmentalized
library, with compartments that can be screened systematically,
e.g., one by one. In other words the invention provides that,
through the selective and judicious use of specific nucleic acid
building blocks, coupled with the selective and judicious use of
sequentially stepped assembly reactions, an experimental design can
be achieved where specific sets of progeny products are made in
each of several reaction vessels. This allows a systematic
examination and screening procedure to be performed. Thus, it
allows a potentially very large number of progeny molecules to be
examined systematically in smaller groups.
[0215] Because of its ability to perform chimerizations in a manner
that is highly flexible yet exhaustive and systematic as well,
particularly when there is a low level of homology among the
progenitor molecules, the instant invention provides for the
generation of a library (or set) comprised of a large number of
progeny molecules. Because of the non-stochastic nature of the
instant ligation reassembly invention, the progeny molecules
generated preferably comprise a library of finalized chimeric
nucleic acid molecules having an overall assembly order that is
chosen by design. In a particularly embodiment, such a generated
library is comprised of greater than 10.sup.3 to greater than
10.sup.1000 different progeny molecular species.
[0216] In one aspect, a set of finalized chimeric nucleic acid
molecules, produced as described is comprised of a polynucleotide
encoding a polypeptide. According to one embodiment, this
polynucleotide is a gene, which may be a man-made gene. According
to another embodiment, this polynucleotide is a gene pathway, which
may be a man-made gene pathway. The invention provides that one or
more man-made genes generated by the invention may be incorporated
into a man-made gene pathway, such as pathway operable in a
eukaryotic organism (including a plant).
[0217] In another exemplification, the synthetic nature of the step
in which the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.,
by mutagenesis) or in an in vivo process (e.g., by utilizing the
gene splicing ability of a host organism). It is appreciated that
in many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a demarcation point.
[0218] Thus, according to another embodiment, the invention
provides that a nucleic acid building block can be used to
introduce an intron. Thus, the invention provides that functional
introns may be introduced into a man-made gene of the invention.
The invention also provides that functional introns may be
introduced into a man-made gene pathway of the invention.
Accordingly, the invention provides for the generation of a
chimeric polynucleotide that is a man-made gene containing one (or
more) artificially introduced intron(s).
[0219] Accordingly, the invention also provides for the generation
of a chimeric polynucleotide that is a man-made gene pathway
containing one (or more) artificially introduced intron(s).
Preferably, the artificially introduced intron(s) are functional in
one or more host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene splicing.
The invention provides a process of producing man-made
intron-containing polynucleotides to be introduced into host
organisms for recombination and/or splicing.
[0220] A man-made gene produced using the invention can also serve
as a substrate for recombination with another nucleic acid.
Likewise, a man-made gene pathway produced using the invention can
also serve as a substrate for recombination with another nucleic
acid. In a preferred instance, the recombination is facilitated by,
or occurs at, areas of homology between the man-made
intron-containing gene and a nucleic acid with serves as a
recombination partner. In a particularly preferred instance, the
recombination partner may also be a nucleic acid generated by the
invention, including a man-made gene or a man-made gene pathway.
Recombination may be facilitated by or may occur at areas of
homology that exist at the one (or more) artificially introduced
intron(s) in the man-made gene.
[0221] The synthetic ligation reassembly method of the invention
utilizes a plurality of nucleic acid building blocks, each of which
preferably has two ligatable ends. The two ligatable ends on each
nucleic acid building block may be two blunt ends (i.e. each having
an overhang of zero nucleotides), or preferably one blunt end and
one overhang, or more preferably still two overhangs.
[0222] An overhang for this purpose may be a 3' overhang or a 5'
overhang. Thus, a nucleic acid building block may have a 3'
overhang or alternatively a 5' overhang or alternatively two 3'
overhangs or alternatively two 5' overhangs. The overall order in
which the nucleic acid building blocks are assembled to form a
finalized chimeric nucleic acid molecule is determined by
purposeful experimental design and is not random.
[0223] According to one preferred embodiment, a nucleic acid
building block is generated by chemical synthesis of two
single-stranded nucleic acids (also referred to as single-stranded
oligos) and contacting them so as to allow them to anneal to form a
double-stranded nucleic acid building block.
[0224] A double-stranded nucleic acid building block can be of
variable size. The sizes of these building blocks can be small or
large. Preferred sizes for building block range from 1 base pair
(not including any overhangs) to 100,000 base pairs (not including
any overhangs). Other preferred size ranges are also provided,
which have lower limits of from 1 bp to 10,000 bp (including every
integer value in between), and upper limits of from 2 bp to 100,
000 bp (including every integer value in between).
[0225] Many methods exist by which a double-stranded nucleic acid
building block can be generated that is serviceable for the
invention; and these are known in the art and can be readily
performed by the skilled artisan.
[0226] According to one embodiment, a double-stranded nucleic acid
building block is generated by first generating two single stranded
nucleic acids and allowing them to anneal to form a double-stranded
nucleic acid building block. The two strands of a double-stranded
nucleic acid building block may be complementary at every
nucleotide apart from any that form an overhang; thus containing no
mismatches, apart from any overhang(s). According to another
embodiment, the two strands of a double-stranded nucleic acid
building block are complementary at fewer than every nucleotide
apart from any that form an overhang. Thus, according to this
embodiment, a double-stranded nucleic acid building block can be
used to introduce codon degeneracy. Preferably the codon degeneracy
is introduced using the site-saturation mutagenesis described
herein, using one or more N,N,G/T cassettes or alternatively using
one or more N,N,N cassettes.
[0227] The in vivo recombination method of the invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide.
[0228] 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.
[0229] The invention provides a method for selecting a subset of
polynucleotides from a starting set of polynucleotides, which
method is based on the ability to discriminate one or more
selectable features (or selection markers) present anywhere in a
working polynucleotide, so as to allow one to perform selection for
(positive selection) and/or against (negative selection) each
selectable polynucleotide. In a one aspect, a method is provided
termed end-selection, which method is based on the use of a
selection marker located in part or entirely in a terminal region
of a selectable polynucleotide, and such a selection marker may be
termed an "end-selection marker".
[0230] End-selection may be based on detection of naturally
occurring sequences or on detection of sequences introduced
experimentally (including by any mutagenesis procedure mentioned
herein and not mentioned herein) or on both, even within the same
polynucleotide. An end-selection marker can be a structural
selection marker or a functional selection marker or both a
structural and a functional selection marker. An end-selection
marker may be comprised of a polynucleotide sequence or of a
polypeptide sequence or of any chemical structure or of any
biological or biochemical tag, including markers that can be
selected using methods based on the detection of radioactivity, of
enzymatic activity, of fluorescence, of any optical feature, of a
magnetic property (e.g., using magnetic beads), of
immunoreactivity, and of hybridization.
[0231] End-selection may be applied in combination with any method
for performing mutagenesis. Such mutagenesis methods include, but
are not limited to, methods described herein (supra and infra).
Such methods include, by way of non-limiting exemplification, any
method that may be referred herein or by others in the art by any
of the following terms: "saturation mutagenesis", "shuffling",
"recombination", "re-assembly", "error-prone PCR", "assembly PCR",
"sexual PCR", "crossover PCR", "oligonucleotide primer-directed
mutagenesis", "recursive (and/or exponential) ensemble mutagenesis
(see Arkin and Youvan, 1992)", "cassette mutagenesis", "in vivo
mutagenesis", and "in vitro mutagenesis". Moreover, end-selection
may be performed on molecules produced by any mutagenesis and/or
amplification method (see, e.g., Arnold, 1993; Caldwell and Joyce,
1992; Stemmer, 1994) following which method it is desirable to
select for (including to screen for the presence of) desirable
progeny molecules.
[0232] In addition, end-selection may be applied to a
polynucleotide apart from any mutagenesis method. In a one
embodiment, end-selection, as provided herein, can be used in order
to facilitate a cloning step, such as a step of ligation to another
polynucleotide (including ligation to a vector). The invention thus
provides for end-selection as a means to facilitate library
construction, selection and/or enrichment for desirable
polynucleotides, and cloning in general.
[0233] In a another embodiment, end-selection can be based on
(positive) selection for a polynucleotide; alternatively
end-selection can be based on (negative) selection against a
polynucleotide; and alternatively still, end-selection can be based
on both (positive) selection for, and on (negative) selection
against, a polynucleotide. End-selection, along with other methods
of selection and/or screening, can be performed in an iterative
fashion, with any combination of like or unlike selection and/or
screening methods and mutagenesis or directed evolution methods,
all of which can be performed in an iterative fashion and in any
order, combination, and permutation. It is also appreciated that
end-selection may also be used to select a polynucleotide in a:
circular (e.g., a plasmid or any other circular vector or any other
polynucleotide that is partly circular), and/or branched, and/or
modified or substituted with any chemical group or moiety.
[0234] In one non-limiting aspect, end-selection of a linear
polynucleotide is performed using a general approach based on the
presence of at least one end-selection marker located at or near a
polynucleotide end or terminus (that can be either a 5' end or a 3'
end). In one particular non-limiting exemplification, end-selection
is based on selection for a specific sequence at or near a terminus
such as, but not limited to, a sequence recognized by an enzyme
that recognizes a polynucleotide sequence. An enzyme that
recognizes and catalyzes a chemical modification of a
polynucleotide is referred to herein as a polynucleotide-acting
enzyme. In a preferred embodiment, polynucleotide-acting enzymes
are exemplified non-exclusively by enzymes with
polynucleotide-cleaving activity, enzymes with
polynucleotide-methylating activity, enzymes with
polynucleotide-ligating activity, and enzymes with a plurality of
distinguishable enzymatic activities (including non-exclusively,
e.g., both polynucleotide-cleaving activity and
polynucleotide-ligating activity).
[0235] It is appreciated that relevant polynucleotide-acting
enzymes include any enzymes identifiable by one skilled in the art
(e.g., commercially available) or that may be developed in the
future, though currently unavailable, that are useful for
generating a ligation compatible end, preferably a sticky end, in a
polynucleotide. It may be preferable to use restriction sites that
are not contained, or alternatively that are not expected to be
contained, or alternatively that are unlikely to be contained
(e.g., when sequence information regarding a working polynucleotide
is incomplete) internally in a polynucleotide to be subjected to
end-selection. It is recognized that methods (e.g., mutagenesis
methods) can be used to remove unwanted internal restriction sites.
It is also appreciated that a partial digestion reaction (i.e. a
digestion reaction that proceeds to partial completion) can be used
to achieve digestion at a recognition site in a terminal region
while sparing a susceptible restriction site that occurs internally
in a polynucleotide and that is recognized by the same enzyme. In
one aspect, partial digest are useful because it is appreciated
that certain enzymes show preferential cleavage of the same
recognition sequence depending on the location and environment in
which the recognition sequence occurs.
[0236] It is also appreciated that protection methods can be used
to selectively protect specified restriction sites (e.g., internal
sites) against unwanted digestion by enzymes that would otherwise
cut a working polypeptide in response to the presence of those
sites; and that such protection methods include modifications such
as methylations and base substitutions (e.g., U instead of T) that
inhibit an unwanted enzyme activity.
[0237] In another embodiment of the invention, a useful
end-selection marker is a terminal sequence that is recognized by a
polynucleotide-acting enzyme that recognizes a specific
polynucleotide sequence. In one aspect of the invention, useful
polynucleotide-acting enzymes also include other enzymes in
addition to classic type II restriction enzymes. According to this
preferred aspect of the invention, useful polynucleotide-acting
enzymes also include gyrases (e.g., topoisomerases), helicases,
recombinases, relaxases, and any enzymes related thereto.
[0238] It is appreciated that, end-selection can be used to
distinguish and separate parental template molecules (e.g., to be
subjected to mutagenesis) from progeny molecules (e.g., generated
by mutagenesis). For example, a first set of primers, lacking in a
topoisomerase I recognition site, can be used to modify the
terminal regions of the parental molecules (e.g., in
polymerase-based amplification). A different second set of primers
(e.g., having a topoisomerase I recognition site) can then be used
to generate mutated progeny molecules (e.g., using any
polynucleotide chimerization method, such as interrupted synthesis,
template-switching polymerase-based amplification, or interrupted
synthesis; or using saturation mutagenesis; or using any other
method for introducing a topoisomerase I recognition site into a
mutagenized progeny molecule) from the amplified template
molecules. The use of topoisomerase I-based end-selection can then
facilitate, not only discernment, but selective topoisomerase
I-based ligation of the desired progeny molecules.
[0239] It is appreciated that an end-selection approach using
topoisomerase-based nicking and ligation has several advantages
over previously available selection methods. In sum, this approach
allows one to achieve direction cloning (including expression
cloning).
[0240] The present method can be used to shuffle, by in vitro
and/or in vivo recombination by any of the disclosed methods, and
in any combination, polynucleotide sequences selected by peptide
display methods, wherein an associated polynucleotide encodes a
displayed peptide which is screened for a phenotype (e.g., for
affinity for a predetermined receptor (ligand).
[0241] 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.
[0242] 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.
[0243] 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 publications WO
91/17271, WO 91/18980, WO 91/19818 and WO 93/08278.
[0244] 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.
[0245] 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.
[0246] The invention also provides a method for shuffling a pool of
polynucleotide sequences identified by the methods of the invention
and selected by affinity screening a library of polysomes
displaying nascent peptides (including single-chain antibodies) for
library members which bind to a predetermined receptor (e.g., a
mammalian proteinaceous receptor such as, for example, a
peptidergic hormone receptor, a cell surface receptor, an
intracellular protein which binds to other protein(s) to form
intracellular protein complexes such as hetero-dimers and the like)
or epitope (e.g., an immobilized protein, glycoprotein,
oligosaccharide, and the like).
[0247] 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).
[0248] 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.
[0249] 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).
[0250] 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.
[0251] 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)).
[0252] 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.
[0253] 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).
[0254] In order to overcome many of the limitations in producing
and identifying high-affinity immunoglobulins through
antigen-stimulated 13 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).
[0255] 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; and Lowman et al., 1991; Lerner et al., 1992;
all 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).
[0256] One particularly advantageous approach has been the use of
so-called single-chain fragment variable (scfv) libraries (Marks et
al., 1992, p. 779; Winter and Milstein, 1991; Clackson et al.,
1991; Marks et al., 1991, p. 581; Chaudhary et al., 1990; Chiswell
et al., 1992; McCafferty et al., 1990; and Huston et al., 1988).
Various embodiments of scfv libraries displayed on bacteriophage
coat proteins have been described.
[0257] 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
(SEQ ID NO:3)) or equivalent linker peptide(s). The linker bridges
the C-terminus of the first V region and N-terminus of the second,
ordered as either VH-linker-VL or VL-linker-VH' In principle, the
scfv binding site can faithfully replicate both the affinity and
specificity of its parent antibody combining site.
[0258] 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).
[0259] 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.
[0260] 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 et al., 1993), as
has error-prone PCR and chemical mutagenesis (Deng et al., 1994).
Riechmann (Riechmann et al., 1993) 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 (Barbas et al., 1992) attempted to
circumvent the problem of limited repertoire sizes resulting from
using biased variable region sequences by randomizing the sequence
in a synthetic CDR region of a human tetanus toxoid-binding
Fab.
[0261] 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
(Barbas et al., 1992) 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.
[0262] 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-virus replication in vitro (Duan et al., 1994), and
intracellular expression of an anti-p21rar, scfv has been shown to
inhibit meiotic maturation of Xenopus oocytes (Biocca et al.,
1993). 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).
[0263] 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 WO 92/03918, WO 93/12227, and WO
94/25585), including hybridomas derived therefrom. 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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).
[0271] The invention of shuffling enables the generation of a vast
library of CDR-variant single-chain antibodies. One way to generate
such antibodies is to insert synthetic CDRs into the single-chain
antibody and/or CDR randomization prior to or concomitant with
shuffling. The sequences of the synthetic CDR cassettes are
selected by referring to known sequence data of human CDR and are
selected in the discretion of the practitioner according to the
following guidelines: synthetic CDRs will have at least 40 percent
positional sequence identity to known CDR sequences, and preferably
will have at least 50 to 70 percent positional sequence identity to
known CDR sequences. 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 (Kabat et al., 1991); the pool (s) of
synthetic CDR sequences are calculated to encode CDR peptide
sequences having at least 40 percent sequence identity to at least
one known naturally-occurring human CDR sequence. 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.
[0272] 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.sup.6 unique CDR sequences are included in
the collection, although occasionally 1.times.10.sup.7 to
1.times.10.sup.8 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, L-selectin), or nonhuman disease associated
macromolecule (e.g., bacterial LPS, virion capsid protein or
envelope glycoprotein) and the like.
[0273] 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.
[0274] 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; Lefkovits,
1997).
[0275] 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.
[0276] 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).
[0277] 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. colilBCCP 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 kemals.
[0278] 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.
[0279] 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.
[0280] The invention is further directed to a method for generating
a selected mutant polynucleotide sequence (or a population of
selected polynucleotide sequences) typically in the form of
amplified and/or cloned polynucleotides, whereby the selected
polynucleotide sequences(s) possess at least one desired phenotypic
characteristic (e.g., encodes a polypeptide, promotes transcription
of linked polynucleotides, binds a protein, and the like) which can
be selected for. 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.
[0281] 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.
[0282] It is an object of the invention to provide a process for
producing hybrid polynucleotides which express a useful hybrid
polypeptide by a series of steps comprising:
[0283] (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;
[0284] (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;
[0285] (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;
[0286] (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;
[0287] (e) optionally repeating steps (c) and (d);
[0288] (f) expressing at least one hybrid polypeptide from said
polynucleotide chain, or chains; and
[0289] (g) screening said at least one hybrid polypeptide for a
useful activity.
[0290] In a one 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.
[0291] 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.
[0292] In another embodiment, clones which are identified as having
a biomolecule or bioactivity of interest may also be sequenced to
identify the DNA sequence encoding a polypeptide (e.g., an enzyme)
or the polypeptide sequence itself having the specified activity,
for example. Thus, in accordance with the present invention it is
possible to isolate and identify: (i) DNA encoding a bioactivity of
interest (e.g., an enzyme having a specified enzyme activity), (ii)
biomolecules (e.g., polynucleotides or enzymes having such activity
(including the amino acid sequence thereof)) and (iii) produce
recombinant biomolecules or bioactivities.
[0293] Suitable clones (e.g., 1-1000 or more clones) from the
library are identified by the methods of the invention and
sequenced using, for example, high through-put sequencing
techniques. The exact method of sequencing is not a limiting factor
of the invention. Any method useful in identifying the sequence of
a particular cloned DNA sequence can be used. In general,
sequencing is an adaptation of the natural process of DNA
replication. Therefore, a template (e.g., the vector) and primer
sequences are used. One general template preparation and sequencing
protocol begins with automated picking of bacterial colonies, each
of which contains a separate DNA clone which will function as a
template for the sequencing reaction. The selected clones are
placed into media, and grown overnight. The DNA templates are then
purified from the cells and suspended in water. After DNA
quantification, high-throughput sequencing is performed using a
sequencers, such as Applied Biosystems, Inc., Prism 377 DNA
Sequencers. The resulting sequence data can then be used in
additional methods, including searching a database or
databases.
[0294] A number of source databases are available that contain
either a nucleic acid sequence and/or a deduced amino acid sequence
for use with the invention in identifying or determining the
activity encoded by a particular polynucleotide sequence. All or a
representative portion of the sequences (e.g., about 100 individual
clones) to be tested are used to search a sequence database (e.g.,
GenBank, PFAM or ProDom), either simultaneously or individually. A
number of different methods of performing such sequence searches
are known in the art. The databases can be specific for a
particular organism or a collection of organisms. For example,
there are databases for the C. elegans, Arabadopsis. sp., M.
genitalium, M. jannaschii, E. coli, H. influenzae, S. cerevisiae
and others. The sequence data of the clone is then aligned to the
sequences in the database or databases using algorithms designed to
measure homology between two or more sequences.
[0295] Such sequence alignment methods include, for example, BLAST
(Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins,
1993), and FASTA (Person & Lipman, 1988). The probe sequence
(e.g., the sequence data from the clone) can be any length, and
will be recognized as homologous based upon a threshold homology
value. The threshold value may be predetermined, although this is
not required. The threshold value can be based upon the particular
polynucleotide length. To align sequences a number of different
procedures can be used. Typically, Smith-Waterman or
Needleman-Wunsch algorithms are used. However, as discussed faster
procedures such as BLAST, FASTA, PSI-BLAST can be used.
[0296] For example, optimal alignment of sequences for aligning a
comparison window may be conducted by the local homology algorithm
of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and
Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981;
Smith et al, J Mol Evol, 1981), by the homology alignment algorithm
of Needleman (Needleman and Wuncsch, 1970), by the search of
similarity method of Pearson (Pearson and Lipman, 1988), by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package
Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,
Wis., or the Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin, 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. The similarity of the two sequence
(i.e., the probe sequence and the database sequence) can then be
predicted.
[0297] Such software matches similar sequences by assigning degrees
of homology to various deletions, substitutions and other
modifications. The terms "homology" and "identity" in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same when compared and aligned for maximum correspondence over
a comparison window or designated region as measured using any
number of sequence comparison algorithms or by manual alignment and
visual inspection.
[0298] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0299] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally
aligned.
[0300] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410
(1990), respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0). The
BLAST algorithm parameters W, T, and X determine the sensitivity
and speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands.
[0301] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Natl. Acad. Sci. USA 90:5873 (1993)). One measure
of similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide sequences would occur by
chance. For example, a nucleic acid is considered similar to a
references sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.2, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0302] Sequence homology means that two polynucleotide sequences
are homologous (i.e., on a nucleotide-by-nucleotide basis) over the
window of comparison. A percentage of sequence identity or homology
is calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
homology. This substantial homology denotes a characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a
sequence having at least 60 percent sequence homology, typically at
least 70 percent homology, often 80 to 90 percent sequence
homology, and most commonly at least 99 percent sequence homology
as compared to a reference sequence of a comparison window of at
least 25-50 nucleotides, wherein the percentage of sequence
homology is calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison.
[0303] Sequences having sufficient homology can the be further
identified by any annotations contained in the database, including,
for example, species and activity information. Accordingly, in a
typical environmental sample, a plurality of nucleic acid sequences
will be obtained, cloned, sequenced and corresponding homologous
sequences from a database identified. This information provides a
profile of the polynucleotides present in the sample, including one
or more features associated with the polynucleotide including the
organism and activity associated with that sequence or any
polypeptide encoded by that sequence based on the database
information. As used herein "fingerprint" or "profile" refers to
the fact that each sample will have associated with it a set of
polynucleotides characteristic of the sample and the environment
from which it was derived. Such a profile can include the amount
and type of sequences present in the sample, as well as information
regarding the potential activities encoded by the polynucleotides
and the organisms from which polynucleotides were derived. This
unique pattern is each sample's profile or fingerprint.
[0304] In some instances it may be desirable to express a
particular cloned polynucleotide sequence once its identity or
activity is determined or a suggested identity or activity is
associated with the polynucleotide. In such instances the desired
clone, if not already cloned into an expression vector, is ligated
downstream of a regulatory control element (e.g., a promoter or
enhancer) and cloned into a suitable host cell. Expression vectors
are commercially available along with corresponding host cells for
use in the invention.
[0305] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, phosmids, bacterial artificial
chromosomes, viral nucleic acid (e.g., vaccinia, 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, yeast, and the like) 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: pQE70, pQE60, pQE-9
(Qiagen), psiX174, pBluescript SK, pBluescript KS, pNH8A, pNH16a,
pNH 118A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTI, pSG
(Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0306] The nucleic acid sequence in the expression vector is
operatively linked to an appropriate expression control sequence(s)
(promoter) to direct mRNA synthesis. Particular named bacterial
promoters include lac, lacZ, T3, T7, gpt, lambda PR, PL 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.
[0307] In addition, the expression Vectors typically contain one or
more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0308] The nucleic acid sequence(s) selected, cloned and sequenced
as hereinabove described can additionally be introduced into a
suitable host to prepare a library which is screened for the
desired biomolecule or bioactivity. The selected nucleic acid is
preferably already in a vector which includes appropriate control
sequences whereby a selected nucleic acid encoding a biomolecule or
bioactivity may be expressed, for detection of the desired
activity. The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or the host cell can be a prokaryotic cell, such as a bacterial
cell. The selection of an appropriate host is deemed to be within
the scope of those skilled in the art from the teachings
herein.
[0309] In some instances it may be desirable to perform an
amplification of the nucleic acid sequence present in a sample or a
particular clone that has been isolated. In this embodiment, the
nucleic acid sequence is amplified by PCR reaction or similar
reaction known to those of skill in the art. Commercially available
amplification kits are available to carry out such amplification
reactions.
[0310] In addition, it is important to recognize that the alignment
algorithms and searchable database can be implemented in computer
hardware, software or a combination thereof. Accordingly, the
isolation, processing and identification of nucleic acid or
polypeptide sequences can be implemented in an automated
system.
[0311] In addition to the sequence based techniques described
above, a number of traditional assay system exist for measuring an
enzymatic activity using multi-well plates. For example, existing
screening technology usually relies on two-dimensional well (e.g.,
96-, 384- and 1536-well) plates. The present invention also
provides a capillary array-based approach of that has numerous
advantages over well-based screening techniques, including the
elimination of the need for fluid dispensers for dispensing fluids
(e.g., reactants) into individual well reservoirs, and the reduced
cost per array (e.g., glass capillaries are reusable) (see, for
example, U.S. patent application Ser. No. 09/444,112, filed Nov.
22, 1999, which is incorporated herein by reference in its
entirety).
[0312] Accordingly, the capillaries, capillary array and systems of
the invention are particularly well suited for screening libraries
for activity or biomolecules of interest including polynucleotides.
The screening for activity may be effected on individual expression
clones or may be initially effected on a mixture of expression
clones to ascertain whether or not the mixture has one or more
specified activities. If the mixture has a specified activity, then
the individual clones may be rescreened for such activity or for a
more specific activity after collection from the capillary
array.
[0313] All headings and subheading used herein are provided for the
convenience of the reader and should not be construed to limit the
invention.
[0314] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a clone" includes a plurality of clones and reference to "the
nucleic acid sequence" generally includes reference to one or more
nucleic acid sequences and equivalents thereof known to those
skilled in the art, and so forth.
[0315] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials
described.
[0316] All publications mentioned herein are incorporated herein by
reference in full for the purpose of describing and disclosing the
databases, proteins, and methodologies, which are described in the
publications which might be used in connection with the described
invention. The publications discussed above and throughout the text
are provided solely for their disclosure prior to the filing date
of the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior invention.
[0317] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Example 1
[0318] DNA isolation. DNA is isolated using the IsoQuick Procedure
as per manufacture's instructions (Orca Research Inc., Bothell,
Wash.). The isolated DNA can optionally be normalized according to
Example 2 (below). Upon isolation, the DNA is sheared by pushing
and pulling the DNA through a 25-gauge double-hub needle and a 1-cc
syringe about 500 times. A small amount is run on a 0.8% agarose
gel to make sure the majority of the DNA is in the desired size
range (about 3-6 kb).
[0319] Blunt-ending DNA. The DNA is blunt-ended by mixing 45 Tl of
10.times. Mung Bean Buffer, 2.0 Tl Mung Bean Nuclease (1050 u/Tl)
and water to a final volume of 405 Tl. The mixture is incubated at
37.degree. C. for 15 minutes. The mixture is phenol; chloroform
extracted, followed by an additional chloroform extraction. One ml
of ice cold ethanol is added to the final extract to precipitate
the DNA. The DNA is precipitated for 10 minutes on ice. The DNA is
removed by centrifugation in a microcentrifuge for 30 minutes. The
pellet is washed with 1 ml of 70% ethanol and repelleted in the
microcentrifuge. Following centrifugation, the DNA is dried and
gently resuspended in 26 Tl of TE buffer.
[0320] Methylation of DNA. The DNA is methylated by mixing 4 Tl of
10.times. EcoRI Methylase Buffer, 0.5 Tl SAM (32 mM), 5.0 Tl EcoRI
Methylase (40 u/Tl) and incubating at 37.degree. C. for 1 hour. In
order to insure blunt ends, the following can be added to the
methylation reaction: 5.0 Tl of 100 mM MgCl.sub.2, 8.0 Tl of dNTP
mix (2.5 mM of each dGTP, dATP, dTTP, dCTP), 4.0 Tl of Klenow (5
u/Tl). The mixture is then incubated at 12.degree. C. for 30
minutes.
[0321] After incubating for 30 minutes 450 Tl 1.times.STE is added.
The mixture is phenol/chloroform extracted once followed by an
additional chloroform extraction. One ml of ice cold ethanol is
added to the final extract to precipitate the DNA. The DNA is
precipitated for 10 minutes on ice. The DNA is removed by
centrifugation in a microcentrifuge for 30 minutes. The pellet is
washed with 1 ml of 70% ethanol, repelleted in the microcentrifuge
and allowed to dry for 10 minutes.
[0322] Ligation. The DNA is ligated by gently resuspending the DNA
in 8 Tl EcoRI adapters (from Stratagene's cDNA Synthesis Kit), 1.0
Tl of 10.times. ligation buffer, 1.0 Tl of 10 mM rATP, 1.0 Tl of T4
DNA Ligase (4 Wu/Tl) and incubating at 4.degree. C. for 2 days. The
ligation reaction is terminated by heating for 30 minutes at
70.degree. C.
[0323] Phosphorylation of adapters. The adapter ends are
phosphorylated by mixing the ligation reaction with 1.0 Tl of
10.times. Ligation Buffer, 2.0 Tl of 10 mM rATP, 6.0 Tl of
H.sub.2O, 1.0 Tl of polynucleotide kinase (PNK), and incubating at
37.degree. C. for 30 minutes. After incubating for 30 minutes, 31
Tl of H.sub.2O and 5 ml of 10.times.STE are added to the reaction
and the sample is size fractionated on a Sephacryl S-500 spin
column. The pooled fractions (1-3) are phenol/chloroform extracted
once, followed by an additional chloroform extraction. The DNA is
precipitated by the addition of ice cold ethanol on ice for 10
minutes. The precipitate is pelleted by centrifugation in a
microcentrifuge at high speed for 30 minutes. The resulting pellet
is washed with 1 ml 70% ethanol, repelleted by centrifugation and
allowed to dry for 10 minutes. The sample is resuspended in 10.5 Tl
TE buffer. The sample is not plated, but is ligated directly to
lambda arms as described above, except 2.5 Tl of DNA and no water
is used.
[0324] Sucrose Gradient (2.2 ml) Size Fractionation. Ligation is
stopped by heating the sample to 65.degree. C. for 10 minutes. The
sample is gently loaded on a 2.2 ml sucrose gradient and
centrifuged in a mini-ultracentrifuged 45 k rpm at 20.degree. C.
for 4 hours (no brake). Fractions are collected by puncturing the
bottom of the gradient tube with a 20-gauge needle and allowing the
sucrose to flow through the needle. The first 20 drops are
collected in a Falcon 2059 tube, and then ten 1-drop fractions
(labeled 1-10) are collected. Each drop is about 60 Tl in volume.
Five Tl of each fraction are run on a 0.8% agarose gel to check the
size. Fractions 1-4 (about 10-1.5 kb) are pooled and, in a separate
tube, fractions 5-7 (about 5-0.5 kb) are pooled. One ml of ice cold
ethanol is added to precipitate the DNA and then placed on ice for
10 minutes. The precipitate is pelleted by centrifugation in a
microcentrifuge at high speed for 30 minutes. The pellets are
washed by resuspending them in 1 ml of 70% ethanol and repelleting
them by centrifugation in a microcentrifuge at high speed for 10
minutes, and then dried. Each pellet is then resuspended in 10 Tl
of TE buffer.
[0325] Test Ligation to Lambda Arms. The assay is plated by
spotting 0.5 Tl of the sample on agarose containing ethidium
bromide along with standards (DNA sample of known concentration) to
get an approximate concentration. The samples are then viewed using
UV light and the estimated concentration is compared to the
standards.
[0326] The following ligation reaction (5 Tl reactions) are
prepared and incubated at 4.degree. C. overnight, as shown in Table
1 below:
1TABLE 1 Lambda 10 X 10 mM arms Insert T4 DNA Sample H.sub.2O
Ligase rATP (ZAP) DNA Ligase Fraction 1-4 0.5 Tl 0.5 Tl 0.5 Tl 1.0
Tl 2.0 Tl 0.5 Tl Fraction 5-7 0.5 Tl 0.5 Tl 0.5 Tl 1.0 Tl 2.0 Tl
0.5 Tl
[0327] Test Package and Plate. The ligation reactions are packaged
following manufacturer's protocol. Packaging reactions are stopped
with 500 Tl SM buffer and pooled with packaging that came from the
same ligation. One Tl of each pooled reaction is titered on an
appropriate host (OD.sub.600=1.0) (XL1-Blue MRF). 200 Tl host (in
MgSO.sub.4) are added to Falcon 2059 tubes, inoculated with 1 Tl
packaged phage and incubated at 37.degree. C. for 15 minutes. About
3 ml of 48.degree. C. top agar (50 ml stock containing 150 Tl IPTG
(0.5 M) and 300 Tl X-GAL (350 mg/ml)) are added and plated on 100
mm plates. The plates are incubated overnight at 37.degree. C.
[0328] Amplification of Libraries (5.0.times.10.sup.5 recombinants
from each library). About 3.0 ml host cells (OD.sub.600=1.0) are
added to two 50 ml conical tubes, inoculated with
2.5.times.10.sup.5 pfu of phage per conical tube, and then
incubated at 37.degree. C. for 20 minutes. Top agar is added to
each tube to a final volume of 45 ml. Each tube is plated across
five 150 mm plates. The plates are incubated at 37.degree. C. for
6-8 hours or until plaques are about pin-head in size. The plates
are overlaid with 8-10 ml SM Buffer and placed at 4.degree. C.
overnight (with gentle rocking if possible).
[0329] Harvest Phage. The phage suspension is recovered by pouring
the SM buffer off each plate into a 50 ml conical tube. About 3 ml
of chloroform are added, shaken vigorously and incubated at room
temperature for 15 minutes. The tubes are centrifuged at 2K rpm for
10 minutes to remove cell debris. The supernatant is poured into a
sterile flask, 500 Tl chloroform are added and stored at 4.degree.
C.
[0330] Titer Amplified Library. Serial dilutions of the harvested
phage are made (for example, 10.sup.-5=1 Tl amplified phage in 1 ml
SM Buffer; 10.sup.-6=1 Tl of the 10.sup.-3 dilution in 1 ml SM
Buffer and the like), and 200 Tl host (in 10 mM MgSO.sub.4) are
added to two tubes. One tube is inoculated with 10 Tl of 10.sup.-6
dilution (10.sup.-5). The other tube is inoculated with 1 Tl of
10.sup.-6 dilution (10.sup.-6), and incubated at 37.degree. C. for
15 minutes.
[0331] About 3 ml of 48.degree. C. top agar (50 ml stock containing
150 Tl IPTG (0.5 M) and 37 Tl X-GAL (350 mg/ml)) are added to each
tube and plated on 100 mm plates. The plates are incubated
overnight at 37.degree. C.
[0332] The ZAP II library is excised to create the pBLUESCRIPT
library according to manufacturer's protocols (Stratagene).
[0333] The DNA library can be transformed into host cells (e.g., E.
coli) to generate an expression library of clones.
Example 2
Normalization
[0334] Prior to library generation, purified DNA can be normalized.
DNA is first fractionated according to the following protocol A
sample composed of genomic DNA is purified on a cesium-chloride
gradient. The cesium chloride (Rf=1.3980) solution is filtered
through a 0.2 Tm filter and 15 ml is loaded into a 35 ml OptiSeal
tube (Beckman) The DNA is added and thoroughly mixed. Ten
micrograms of bis-benzimide (Sigma; Hoechst 33258) is added and
mixed thoroughly. The tube is then filled with the filtered cesium
chloride solution and spun in a Bti50 rotor in a Beckman L8-70
Ultracentrifuge at 33 k rpm for 72 hours. Following centrifugation,
a syringe pump and fractionator (Brandel Model 186) are used to
drive the gradient through an ISCO UA-5UV absorbance detector set
to 280 nm. Peaks representing the DNA from the organisms present in
an environmental sample are obtained. Eubacterial sequences can be
detected by PCR amplification of DNA encoding rRNA from a 10 fold
dilution of the E. coli peak using the following primers to
amplify:
2 Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:4) Reverse
primer: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO:5)
[0335] Recovered DNA is sheared or enzymatically digested to 3-6 kb
fragments. Lone-linker primers are ligated and the DNA is
size-selected. Size-selected DNA is amplified by PCR, if
necessary.
[0336] Normalization is then accomplished by resuspending the
double-stranded DNA sample in hybridization buffer (0.12 M
NaH.sub.2PO.sub.4, pH 6.8/0.82 M NaCl/1 mM EDTA/0.1% SDS). The
sample is overlaid with mineral oil and denatured by boiling for 10
minutes. The sample is incubated at 68.degree. C. for 12-36 hours.
Double-stranded DNA is separated from single-stranded DNA according
to standard protocols (Sambrook, 1989) on hydroxyapatite at
60.degree. C. The single-stranded DNA fraction is desalted and
amplified by PCR. The process is repeated for several more rounds
(up to 5 or more).
Example 3
Enzymatic Activity Assay
[0337] The following is a representative example of a procedure for
screening an expression library, prepared in accordance with
Example 1, for hydrolase activity.
[0338] Plates of the library prepared as described in Example 1 are
used to multiply inoculate a single plate containing 200 Tl of LB
Amp/Meth, glycerol in each well. This step is performed using the
High Density Replicating Tool (HDRT) of the Beckman BIOMEK.RTM.
with a 1% bleach, water, isopropanol, air-dry sterilization cycle
between each inoculation. The single plate is grown for 2 h at
37.degree. C. and is then used to inoculate two white 96-well
Dynatech microtiter daughter plates containing 250 Tl of LB
Amp/Meth, glycerol in each well. The original single plate is
incubated at 37.degree. C. for 18 h, then stored at -80.degree. C.
The two condensed daughter plates are incubated at 37.degree. C.
also for 18 h. The condensed daughter plates are then heated at
70.degree. C. for 45 min. to kill the cells and inactivate the host
E. coli enzymes. A stock solution of 5 mg/mL morphourea
phenylalanyl-7-amino-4-trifluoromethyl coumarin (MuPheAFC, the
"substrate") in DMSO is diluted to 600 TM with 50 mM pH 7.5 Hepes
buffer containing 0.6 mg/mL of the detergent dodecyl maltoside.
Fifty Tl of the 600 TM MuPheAFC solution is added to each of the
wells of the white condensed plates with one 100 Tl mix cycle using
the BIOMEK to yield a final concentration of substrate of about 100
TM. The fluorescence values are recorded (excitation=400 nm,
emission=505 nm) on a plate reading fluorometer immediately after
addition of the substrate (t=0). The plate is incubated at
70.degree. C. for 100 min, then allowed to cool to ambient
temperature for 15 additional minutes. The fluorescence values are
recorded again 1
[0339] (t=100). The values at t=0 are subtracted from the values at
t=100 to determine if an active clone is present.
MuPheAFC
[0340] The data will indicate whether one of the clones in a
particular well is hydrolyzing the substrate. In order to determine
the individual clone which carries the activity, the source library
plates are thawed and the individual clones are used to singly
inoculate a new plate containing LB Amp/Meth, glycerol. As above,
the plate is incubated at 37.degree. C. to grow the cells, heated
at 70.degree. C. to inactivate the host enzymes, and 50 Tl of 600
TM MuPheAFC is added using the Biomek.
[0341] After addition of the substrate the t=0 fluorescence values
are recorded, the plate is incubated at 70.degree. C., and the
t=100 min. values are recorded as above. These data indicate which
plate the active clone is in.
[0342] The enantioselectivity value, E, for the substrate is
determined according to the equation below: 1 E = ln [ ( 1 - c ( 1
+ ee p ) ] ln [ ( 1 - c ( 1 + ee p ) ]
[0343] where ee.sub.p=the enantiomeric excess (ee) of the
hydrolyzed product and c=the percent conversion of the reaction.
See Wong and Whitesides, Enzymes in Synthetic Organic Chemistry,
1994, Elsevier, Tarrytown, N.Y., pp. 9-12.
[0344] The enantiomeric excess is determined by either chiral high
performance liquid chromatography (HPLC) or chiral capillary
electrophoresis (CE). Assays are performed as follows: two hundred
Tl of the appropriate buffer is added to each well of a 96-well
white microtiter plate, followed by 50 Tl of partially or
completely purified enzyme solution; 50 Tl of substrate is added
and the increase in fluorescence monitored versus time until 50% of
the substrate is consumed or the reaction stops, whichever comes
first.
Example 4
Directed Mutagenesis of Positive Enzyme Activity Clones
[0345] Directed mutagenesis was performed on two different enzymes
(alkaline phosphatase and .theta.-glycosidase) to generate new
enzymes which exhibit a higher degree of activity than the
wild-type enzymes.
[0346] Alkaline Phosphatase
[0347] The XL1-Red strain (Stratagene) was transformed with genomic
clone 27a3a (in plasmid pBluescript) encoding the alkaline
phosphatase gene from the organism OC9a, an organism isolated from
the surface of a whale bone, according to the manufacturer's
protocol. A 5 ml culture of LB+0.1 mg/ml ampicillin was inoculated
with 200 Tl of the transformation and the culture was allowed to
grow at 37.degree. C. for 30 hours. A miniprep was then performed
on the culture, and the isolated DNA screened by transforming 2 Tl
of the resulting DNA into XL-1 Blue cells (Stratagene) according to
the manufacturer's protocol and following the assay procedure
outlined below. The mutated OC9a phosphatase took 10 minutes to
develop color and the wild type enzyme took 30 minutes to develop
color in the screening assay.
[0348] Standard Alkaline Phosphatase Screening Assay
[0349] Transformed XL1 Blue cells were plated on LB/amp plates. The
resulting colonies were lifted with Duralon UV (Stratagene) or HATF
(Millipore) membranes and lysed in chloroform vapors for 30
seconds. Cells were heat killed by incubating for 30 minutes at
85.degree. C. The filters were developed at room temperature in
BCIP buffer and the fastest developing colonies ("positives") were
selected for restreaking the "positives" onto a BCIP plate (BCIP
Buffer: 20 mm CAPS pH 9.0, 1 mm MgCl.sub.2, 0.01 mm ZnCl.sub.2, 0.1
mg/ml BCIP).
[0350] Beta-Glycosidase
[0351] This protocol was used to mutagenize Thermococcus 9N2
Beta-Glycosidase.
[0352] PCR was carried out by incubating 2 microliters dNTP's (10
mM Stocks); 10 microliters 10.times.PCR Buffer; 0.5 microliters
Vector DNA-31G1A-100 nanograms; 20 microliters 3' Primer (100
pmol); 20 microliters 5' Primer (100 pmol); 16 microliters MnCl
4H.sub.2O (1.25 mM Stock); 24.5 microliters H.sub.2O; and 1
microliter Taq Polymerase (5.0 Units) in a total volume of 100
microliters. The PCR cycle was: 95.degree. C. 15 seconds;
58.degree. C. 30 seconds; 72.degree. C. 90 seconds; 25 cycles (10
minute extension at 72.degree. C.-4.degree. C. incubation).
[0353] Five microliters of the PCR product was run on a 1% agarose
gel to check the reaction. Purify on a QIAQUICK column (Qiagen).
Resuspend in 50 microliters H.sub.2O.
[0354] Twenty-five microliters of purified PCR product; 10
microliters NEB Buffer #2; 3 microliters Kpn I (10U/microliter); 3
microliters EcoR1 (20 U/microliter); and 59 microliters H.sub.2O.
were incubated for 2 hours at 37.degree. C. to digest the PCR
products and purified on a QIAQUICK column (Qiagen). Elute with 35
microliters H.sub.2O.
[0355] Ten microliters of digested PCR product, 5 microliters
Vector (cut with EcoRI/KpnI and phosphatased with shrimp alkaline
phosphatase, 4 microliters 5.times. Ligation Buffer, and 1
microliter T4 DNA Ligase (BRL) were incubated overnight to ligate
the PCR products into the vector.
[0356] The resulting vector was transformed into M15pREP4 cells
using electroporation. 100 or 200 microliters of the cells were
plated onto LB amp meth kan plates, and grown overnight at
37.degree. C.
[0357] Beta-galactosidase was assayed by (1) Perform colony lifts
using Millipore HATF membrane filters; (2) lyse colonies with
chloroform vapor in 150 mm glass petri dishes; (3) transfer filters
to 100 mm glass petri dishes containing a piece of Whatman 3MM
filter paper saturated with Z buffer containing 1 mg/ml XGLU (After
transferring filter bearing lysed colonies to the glass petri dish,
maintain dish at room temperature); and (4) "Positives" were
observed as blue spots on the filter membranes ("positives" are
spots which appear early). A pasteur pipette (or glass capillary
tube) was used to core blue spots on the filter membrane. Place the
small filter disk in an Eppendorf tube containing 20Tl water.
Incubate the Eppendorf tube at 75.degree. C. for 5 minutes followed
by vortexing to elute plasmid DNA off filter. Transform this DNA
into electrocompetent E. coli cells and repeat filter-lift assay on
transformation plates to identify "positives." Return
transformation plates to 37.degree. C. incubator after filter lift
to regenerate colonies. Inoculate 3 ml LBamp liquid with repurified
positives and incubate at 37.degree. C. overnight. Isolate plasmid
DNA from these cultures and sequence plasmid insert. The filter
assay uses buffer Z (see recipe below) containing 1 mg/ml of the
substrate 5-bromo-4-chloro-3-indo- lyl-.beta.-o-glucopyranoside
(XGLU) (Diagnostic Chemicals Limited or Sigma). Z-Buffer:
(referenced in Miller, J. H. (1992) A Short Course in Bacterial
Genetics, p. 445.) per liter:
3 Na.sub.2HPO.sub.4-7H.sub.2O 16.1 g Na.sub.2HPO.sub.4-4H.sub.2O
5.5 g KCl 0.75 g Na.sub.2HPO.sub.4-7H.sub.2O 0.246 g
6-mercaptoethanol 2.7 ml Adjust pH to 7.0
Example 5
Construction of a Stable, Large Insert DNA Library of Picoplankton
Genomic DNA
[0358] Cell collection and preparation of DNA. Agarose plugs
containing concentrated picoplankton cells were prepared from
samples collected on an oceanographic cruise from Newport, Oreg. to
Honolulu, Hi. Seawater (30 liters) was collected in Niskin bottles,
screened through 10 Tm Nitex, and concentrated by hollow fiber
filtration (Amicon DC 10) through 30,000 MW cutoff polyfulfone
filters. The concentrated bacterioplankton cells were collected on
a 0.22 Tm, 47 mm Durapore filter, and resuspended in 1 ml of
2.times.STE buffer (1 M NaCl, 0.1M EDTA, 10 mM Tris, pH 8.0) to a
final density of approximately 1.times.10.sup.10 cells per ml. The
cell suspension was mixed with one volume of 1% molten Seaplaque
LMP agarose (FMC) cooled to 40.degree. C., and then immediately
drawn into a 1 ml syringe. The syringe was sealed with parafilm and
placed on ice for 10 min. The cell-containing agarose plug was
extruded into 10 ml of Lysis Buffer (10 mM Tris pH 8.0, 50 mM NaCl,
0.1M EDTA, 1% Sarkosyl, 0.2% sodium deoxycholate, 1 mg/ml lysozyme)
and incubated at 37.degree. C. for one hour. The agarose plug was
then transferred to 40 mls of ESP Buffer (1% Sarkosyl, 1 mg/ml
proteinase K, in 0.5M EDTA), and incubated at 55.degree. C. for 16
hours. The solution was decanted and replaced with fresh ESP
Buffer, and incubated at 55.degree. C. for an additional hour. The
agarose plugs were then placed in 50 mM EDTA and stored at
4.degree. C. shipboard for the duration of the oceanographic
cruise.
[0359] One slice of an agarose plug (72 Tl) prepared from a sample
collected off the Oregon coast was dialyzed overnight at 4.degree.
C. against 1 mL of buffer A (100 mM NaCl, 10 mM Bis Tris
Propane-HCl, 100 Tg/ml acetylated BSA: pH 7.0 at 25.degree. C.) in
a 2 mL microcentrifuge tube. The solution was replaced with 250 Tl
of fresh buffer A containing 10 mM MgCl.sub.2 and 1 mM DTT and
incubated on a rocking platform for 1 hr at room temperature. The
solution was then changed to 250 Tl of the same buffer containing
4U of Sau3Al (NEB), equilibrated to 37.degree. C. in a water bath,
and then incubated on a rocking platform in a 37.degree. C.
incubator for 45 min. The plug was transferred to a 1.5 ml
microcentrifuge tube and incubated at 68.degree. C. for 30 min to
inactivate the enzyme and to melt the agarose. The agarose was
digested and the DNA dephosphorylased using Gelase and
HK-phosphatase (Epicentre), respectively, according to the
manufacturer's recommendations. Protein was removed by gentle
phenol/chloroform extraction and the DNA was ethanol precipitated,
pelleted, and then washed with 70% ethanol. This partially digested
DNA was resuspended in sterile H.sub.2O to a concentration of 2.5
ng/Tl for ligation to the pFOS1 vector.
[0360] PCR amplification results from several of the agarose plugs
indicated the presence of significant amounts of archaeal DNA.
Quantitative hybridization experiments using rRNA extracted from
one sample, collected at 200 m of depth off the Oregon Coast,
indicated that planktonic archaea in (this assemblage comprised
approximately 4.7% of the total picoplankton biomass (this sample
corresponds to "PACI"-200 m in Table I of DeLong et al., Nature,
371:695-698, 1994). Results from archaeal-biased rDNA PCR
amplification performed on agarose plug lysates confirmed the
presence of relatively large amounts of archaeal DNA in this
sample. Agarose plugs prepared from this picoplankton sample were
chosen for subsequent fosmid library preparation. Each 1 ml agarose
plug from this site contained approximately 7.5.times.10.sup.5
cells, therefore approximately 5.4.times.10.sup.5 cells were
present in the 72 Tl slice used in the preparation of the partially
digested DNA.
[0361] Vector arms were prepared from pFOS1 as described (Kim et
al., Stable propagation of cosmid sized human DNA inserts in an F
factor based vector, Nucl. Acids Res., 20:10832-10835, 1992).
Briefly, the plasmid was completely digested with AstII,
dephosphorylated with HK phosphatase, and then digested with BamHI
to generate two arms, each of which contained a cos site in the
proper orientation for cloning and packaging ligated DNA between
35-45 kbp. The partially digested picoplankton DNA was ligated
overnight to the PFOS1 arms in a 15 Tl ligation reaction containing
25 ng each of vector and insert and 1U of T4 DNA ligase
(Boehringer-Mannheim). The ligated DNA in four microliters of this
reaction was in vitro packaged using the Gigapack XL packaging
system (Stratagene), the fosmid particles transfected to E. coli
strain DH10B (BRL), and the cells spread onto LB.sub.cm15 plates.
The resultant fosmid clones were picked into 96-well microliter
dishes containing LB.sub.cm15 supplemented with 7% glycerol.
Recombinant fosmids, each containing ca. 40 kb of picoplankton DNA
insert, yielded a library of 3.552 fosmid clones, containing
approximately 1.4.times.10.sup.8 base pairs of cloned DNA. All of
the clones examined contained inserts ranging from 38 to 42 kbp.
This library was stored frozen at -80.degree. C. for later
analysis.
[0362] Numerous modifications and variations of the present
invention are possible in light of the above teachings; therefore,
within the scope of the claims, the invention may be practiced
other than as particularly described. Whilec the invention has been
described in detail with reference to certain preferred embodiments
thereof, it will be understood that modifications and variations
are within the spirit and scope of that which is described and
claimed.
* * * * *
References