U.S. patent application number 12/802591 was filed with the patent office on 2011-03-10 for polymerases for nucleotide analogue incorporation.
This patent application is currently assigned to Pacific Biosciences of California, Inc.. Invention is credited to Arek Bibillo, Fred Christians, Sonya Clark, David K. Hanzel, John Lyle, Paul Mitsis, Devon Murphy, Geoff Otto, Insil Park, Paul Peluso, Thang Pham, David R. Rank.
Application Number | 20110059505 12/802591 |
Document ID | / |
Family ID | 38218682 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110059505 |
Kind Code |
A1 |
Hanzel; David K. ; et
al. |
March 10, 2011 |
Polymerases for nucleotide analogue incorporation
Abstract
Compositions that include polymerases with features for
improving entry of nucleotide analogues into active site regions
and for coordinating with the nucleotide analogues in the active
site region are provided. Methods of making the polymerases and of
using the polymerases in sequencing and DNA replication and
amplification as well as kinetic models of polymerase activity and
computer-implemented methods of using the models are also
provided.
Inventors: |
Hanzel; David K.; (Palo
Alto, CA) ; Otto; Geoff; (San Carlos, CA) ;
Murphy; Devon; (Mountain View, CA) ; Peluso;
Paul; (San Carlos, CA) ; Pham; Thang;
(Mountain View, CA) ; Rank; David R.; (Pacific
Grove, CA) ; Mitsis; Paul; (Trenton, NJ) ;
Christians; Fred; (Los Altos Hills, CA) ; Bibillo;
Arek; (Cupertino, CA) ; Park; Insil; (Fremont,
CA) ; Clark; Sonya; (Oakland, CA) ; Lyle;
John; (Redwood Shores, CA) |
Assignee: |
Pacific Biosciences of California,
Inc.
|
Family ID: |
38218682 |
Appl. No.: |
12/802591 |
Filed: |
June 8, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11645223 |
Dec 21, 2006 |
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12802591 |
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60753670 |
Dec 22, 2005 |
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Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C12P 19/34 20130101; C07K 2319/20 20130101; C12Q 1/6869 20130101;
Y02P 20/52 20151101; C12N 9/1252 20130101; C12Y 207/07007
20130101 |
Class at
Publication: |
435/194 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Portions of the invention were made with government support
under NHGR1 Grant No. R01 HG003710-01, and the government may have
certain rights to the invention.
Claims
1-55. (canceled)
56. A recombinant nucleic acid polymerase comprising at least a
first exogenous tag at the C-terminal region of the polymerase and
at least a second exogenous tag at the N-terminal region of the
polymerase.
57. The polymerase of claim 56, wherein the first tag is selected
from: a polyhistidine tag, a HIS-6 tag, a GST tag, a BiTag, an S
Tag, a SNAP-tag, an HA tag, a plurality of HIS-6 tags, a plurality
of GST tags, a plurality BiTags, a plurality of S Tags, a plurality
of SNAP-tags, a plurality of HA tags, biotin, avidin, one or more.
enterokinase sites, thrombin sites, antibodies or antibody domains,
antibody fragments, antigens, receptors, receptor domains, receptor
fragments, ligands, dyes, acceptors, quenchers, or combinations
thereof.
58. The polymerase of claim 56, wherein the second tag is selected
from: a polyhistidine tag, a HIS-6 tag, a GST tag, a BiTag, an S
Tag, a SNAP-tag, an HA tag, a plurality of HIS-6 tags, a plurality
of GST tags, a plurality BiTags, a plurality of S Tags, a plurality
of SNAP-tags, a plurality of HA tags, biotin, avidin, one or more
enterokinase sites, thrombin sites, antibodies or antibody domains,
antibody fragments, antigens, receptors, receptor domains, receptor
fragments, ligands, dyes, acceptors, quenchers, or combinations
thereof.
59. The polymerase of claim 56, wherein the first tag and the
second tag are the same.
60. The polymerase of claim 59, wherein the first and second tags
are polyhistidine tags.
61. The polymerase of claim 56, wherein the first and second tags
are different.
62. The polymerase of claim 56, comprising a plurality of tags at
the C-terminal region or a plurality of tags at the N-terminal
region, or both.
63. The polymerase of claim 56, wherein the polymerase is
homologous to a 129 DNA polymerase.
64. The polymerase of claim 56, wherein the tag is selected from:
an affinity tag, a purification tag and a substrate binding
tag.
65. The polymerase of claim 64, wherein the substrate binding tag
is capable of binding biotin or avidin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 11/645,223,
filed Dec. 21, 2006, entitled "POLYMERASES FOR NUCLEOTIDE ANALOGUE
INCORPORATION" by David K. Hanzel, et al., which claims priority to
and benefit of the following prior provisional patent application:
U.S. Ser. No. 60/753,670, filed Dec. 22, 2005, entitled
"POLYMERASES FOR NUCLEOTIDE ANALOGUE INCORPORATION" by David K.
Hanzel et al. Each of these applications is incorporated herein by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0003] The invention relates to polymerases with features for
improving entry of nucleotide analogues into active site regions
and for coordinating with the nucleotide analogues in the active
site region. Methods of making the polymerases and of using the
polymerases in sequencing and DNA replication and amplification, as
well as kinetic models of polymerase activity and
computer-implemented methods of using the models, are also
described.
BACKGROUND OF THE INVENTION
[0004] DNA polymerases replicate the genomes of living organisms.
In addition to this central role in biology, DNA polymerases are
also ubiquitous tools of biotechnology. They are widely used, e.g.,
for reverse transcription, amplification, labeling, and sequencing,
which are central technologies for a variety of applications such
as sequencing, nucleic acid amplification, cloning, protein
engineering, diagnostics, molecular medicine and many other
technologies.
[0005] Because of the significance of DNA polymerases, they have
been extensively studied. This study has focused, e.g., on
phylogenetic relationships among polymerases, structure of
polymerases, structure-function features of polymerases, and the
role of polymerases in DNA replication and other basic biology, as
well as ways of using DNA polymerases in biotechnology. For a
review of polymerases, see, e.g., Hubscher et al. (2002) EUKARYOTIC
DNA POLYMERASES Annual Review of Biochemistry Vol. 71: 133-163;
Alba (2001) "Protein Family Review: Replicative DNA Polymerases"
Genome Biology 2(1): reviews 3002.1-3002.4; Steitz (1999) "DNA
polymerases: structural diversity and common mechanisms" J Biol
Chem 274:17395-17398 and Burgers et al. (2001) "Eukaryotic DNA
polymerases: proposal for a revised nomenclature" J Biol Chem.
276(47):43487-90. Crystal structures have been solved for many
polymerases, which often share a similar architecture. The basic
mechanisms of action for many polymerases have been determined.
[0006] A fundamental application of DNA technology involves various
labeling strategies for labeling a DNA that is produced by a DNA
polymerase. This is useful in microarray technology, DNA
sequencing, SNP detection, cloning, PCR analysis, and many other
applications. Labeling is often performed in various post-synthesis
hybridization or chemical labeling schemes, but DNA polymerases
have also been used to directly incorporate various labeled
nucleotides in a variety of applications, e.g., via nick
translation, reverse transcription, random priming, amplification,
the polymerase chain reaction, etc. See, e.g., Giller et al. (2003)
"Incorporation of reporter molecule-labeled nucleotides by DNA
polymerases. I. Chemical synthesis of various reporter
group-labeled 2'-deoxyribonucleoside-5'-triphosphates" Nucleic
Acids Res. 31(10): 2630-2635; Augustin et al. (2001) "Progress
towards single-molecule sequencing: enzymatic synthesis of
nucleotide-specifically labeled DNA" J. Biotechnol., 86:289-301;
Tonon et al. (2000) "Spectral karyotyping combined with
locus-specific FISH simultaneously defines genes and chromosomes
involved in chromosomal translocations" Genes Chromosom. Cancer
27:418-423; Zhu and Waggoner (1997) "Molecular mechanism
controlling the incorporation of fluorescent nucleotides into DNA
by PCR." Cytometry, 28:206-211. Yu et al. (1994) "Cyanine dye dUTP
analogs for enzymatic labeling of DNA probes" Nucleic Acids Res.,
22:3226-3232; Zhu et al. (1994) "Directly labeled DNA probes using
fluorescent nucleotides with different length linkers." Nucleic
Acids Res. 22:3418-3422; Ried et al. (1992) "Simultaneous
visualization of seven different DNA probes by in situ
hybridization using combinatorial fluorescence and digital imaging
microscopy" Proc. Natl Acad. Sci. USA, 89:1388-1392.
[0007] DNA polymerase mutants have been identified that have
altered nucleotide analogue incorporation properties relative to
wild-type counterpart enzymes. For example, Vent.sup.A488L DNA
polymerase can incorporate certain non-standard nucleotides with a
higher efficiency than native Vent DNA polymerase. See Gardner et
al. (2004) "Comparative Kinetics of Nucleotide Analog Incorporation
by Vent DNA Polymerase" J. Biol. Chem., 279(12), 11834-11842;
Gardner and Jack "Determinants of nucleotide sugar recognition in
an archaeon DNA polymerase" Nucleic Acids Research, 27(12)
2545-2553. The altered residue in this mutant, A488, is predicted
to be facing away from the nucleotide binding site of the enzyme.
The pattern of relaxed specificity at this position roughly
correlates with the size of the substituted amino acid side chain
and affects incorporation by the enzyme of a variety of modified
nucleotide sugars.
[0008] The ability to improve specificity, processivity, or other
features of DNA polymerases towards labeled nucleotide analogues
would be highly desirable in a variety of contexts where, e.g.,
nucleic acid labeling is desired, including DNA amplification,
sequencing, labeling, detection, cloning, and many others. The
present invention provides new DNA polymerases with modified
properties for labeled nucleotide analogues, methods of making such
polymerases, methods of using such polymerases, and many other
features that will become apparent upon a complete review of the
following.
SUMMARY OF THE INVENTION
[0009] The invention includes polymerases that incorporate
nucleotide analogues, such as phosphate analogues, into a growing
template copy, during DNA amplification. Without being bound to any
particular theory of operation, these polymerases are optionally
modified such that the active site of the polymerase is modified to
reduce steric entry inhibition of the analogue into the active site
and/or to provide complementarity with one or more non-natural
features of the nucleotide analogue. Such polymerases are
particularly well-suited for DNA amplification and/or sequencing
applications, including real-time applications, e.g., in the
context of amplification or sequencing protocols that include
incorporation of analogue residues into DNA by the polymerase. The
analogue residue that is incorporated can be the same as a natural
residue, e.g., where a label or other moiety of the analogue is
removed by action of the polymerase during incorporation, or the
analogue residue can have one or more feature that distinguishes it
from a natural nucleotide residue.
[0010] Accordingly, the invention includes compositions that
include a recombinant DNA polymerase. The recombinant DNA
polymerase includes a modified active site region that is
homologous to a wild-type active site region of a wild-type DNA
polymerase. The modified active site region includes one or more
structural modifications relative to the wild type active site
region that improve the desired activity of the enzyme, e.g.,
toward naturally occurring nucleotides and/or nucleotide analogues.
In certain aspects, and without being bound to a particular theory
of operation, such modifications include those that reduce steric
inhibition for entry of a natural nucleotide or nucleotide analogue
into the modified active site region and/or that make the active
site region complementary with one or more non-natural features of
the natural nucleotide and/or nucleotide analogue. The recombinant
DNA polymerase displays a modified property for the nucleotide
analogue as compared to the wild-type polymerase.
[0011] A variety of DNA polymerases are optionally modified to
include the modified active site region. For example, the
recombinant DNA polymerase is optionally homologous to a .PHI.29
DNA polymerase or mutant thereof, a Taq polymerase, an exonuclease
deficient Taq polymerase, a DNA Pol I polymerase, a T7 polymerase,
an RB69 polymerase, a T5 polymerase, or a polymerase corresponding
to a Klenow fragment of a DNA Pol I polymerase. For example, the
recombinant DNA polymerase can be homologous to a wild-type or
exonuclease deficient .PHI.29 DNA polymerase, e.g., as described in
U.S. Pat. No. 5,001,050, 5,198,543, or 5,576,204. Similarly, the
recombinant DNA polymerase can be homologous to .PHI.29, B103,
GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5,
Cp-7, PR4, PR5, PR722, or L17, or the like. For nomenclature, see
also, Meijer et al. (2001) ".PHI.29 Family of Phages" Microbiology
and Molecular Biology Reviews, 65(2):261-287.
[0012] The modified active site region can include any of a variety
of different modifications to reduce steric inhibition and/or to
make the region complementary with one or more non-natural features
of the nucleotide analogue. For example, structural modifications
within or proximal to the active site relative to the wild-type
.PHI.29 DNA polymerase are selected from: a .DELTA.505-525
deletion, a deletion within .DELTA.505-525, a K135A mutation, an
L384R mutation in combination with another mutation herein (when an
L384R mutation is present, it will generally be in combination with
one or more additional mutation that reduces steric inhibition for
entry of the nucleotide analogue), an E375H mutation, an E375S
mutation, an E375K mutation, an E375R mutation, an E375A mutation,
an E375Q mutation, an E375W mutation, an E375Y mutation, an E375F
mutation, an E486A mutation, an E486D mutation, a K512A mutation,
and combinations thereof. The polymerase can also include an
additional mutation or combination of mutations selected from those
listed in Table 8.
[0013] The polymerase optionally further includes one or more
mutations/deletions relative to the wild-type polymerase that
reduce or eliminate endogenous exonuclease activity. For example,
relative to the wild-type .PHI.29 DNA polymerase, N62 is optionally
mutated or deleted to reduce exonuclease activity; e.g., the
polymerase can include an N62D mutation. Other example mutations
that reduce exonuclease activity include: D12A, T15I, E141, and/or
D66A; accordingly, the polymerases of the invention optionally
comprise one or more of these mutations.
[0014] The recombinant DNA polymerase optionally includes
additional features exogenous or heterologous to a corresponding
DNA polymerase such as a wild-type or nuclease deficient
polymerase. For example, the recombinant polymerase optionally
includes one or more exogenous affinity tags, e.g., purification or
substrate binding tags, such as a 6 His tag sequence, a GST tag, an
HA tag sequence, a plurality of 6 His tag sequences, a plurality of
GST tags, a plurality of HA tag sequences, a SNAP-tag, or the like.
These may be inserted into any of a variety of positions within the
protein, and are preferably at one or more termini, e.g., C
terminus or N terminus of the protein, and are more preferably, at
the terminus that is most distal to the active site in the 3D
structure of the protein.
[0015] Example polymerases of the invention include those listed in
Table 3.
[0016] The compositions optionally include the nucleotide analogue.
Example nucleotide analogues include those that include fluorophore
and/or dye moieties. For example, the nucleotide analogue can be a
labeled nucleotide, e.g., a base, sugar and/or phosphate labeled
nucleotide. The analogue can be a mono-deoxy or a dideoxy
nucleotide analogue.
[0017] One example class of nucleotide analogues are
phosphate-labeled nucleotide analogues, including mono-deoxy
phosphate-labeled nucleotide analogues and/or dideoxy
phosphate-labeled nucleotide analogues. For example, the nucleotide
analogue can be a labeled nucleotide analogue having from 3 to 6
phosphate groups (e.g., where the nucleotide analogue is a
triphosphate, a tetraphosphate, a pentaphosphate or a
hexaphosphate).
[0018] For example, the composition can include a labeled compound
of the formula:
##STR00001##
wherein B is a nucleobase (note that B optionally includes a
label); S is selected from a sugar moiety, an acyclic moiety or a
carbocyclic moiety (note that S optionally includes a label); L is
an optional detectable label; R.sub.1 is selected from O and S;
R.sub.2, R.sub.3 and R.sub.4 are independently selected from O, NH,
S, methylene, substituted methylene, C(O), C(CH.sub.2), CNH.sub.2,
CH.sub.2CH.sub.2, C(OH)CH.sub.2R where R is 4-pyridine or
1-imidazole, provided that R.sub.4 may additionally be selected
from
##STR00002##
R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.11 and R.sub.13 are, when
present, each independently selected from O, BH.sub.3, and S; and
R.sub.9, R.sub.10 and R.sub.12 are independently selected from O,
NH, S, methylene, substituted methylene, CNH.sub.2,
CH.sub.2CH.sub.2, C(OH)CH.sub.2R where R is 4-pyridine or
1-imidazole. In some cases, phosphonate analogs may be employed as
the analogs, e.g., where one of R.sub.2, R.sub.3, R.sub.4, R.sub.9,
R.sub.10 or R.sub.12 are not O, e.g., they are methyl etc.
[0019] The recombinant DNA polymerase displays a modified property
for the nucleotide analogue as compared to the wild-type
polymerase. For example, the modified property can be, e.g.,
K.sub.m, k.sub.cat, V.sub.max, recombinant polymerase processivity
in the presence of the nucleotide analogue (or of a naturally
occurring nucleotide), average template read-length by the
recombinant polymerase in the presence of the nucleotide analogue,
specificity of the recombinant polymerase for the nucleotide
analogue, rate of binding of the nucleotide analogue, rate of
product (pyrophosphate, triphosphate, etc.) release, and/or
branching rate. In one desirable embodiment, the modified property
is a reduced K.sub.m for the nucleotide analogue and/or an
increased k.sub.cat/K.sub.m or V.sub.max/K.sub.m for the nucleotide
analogue. Similarly, the recombinant polymerase optionally has an
increased rate of binding of the nucleotide analogue, an increased
rate of product release, and/or a decreased branching rate, as
compared to the wild-type polymerase.
[0020] At the same time, the recombinant DNA polymerase can
incorporate natural nucleotides (e.g., A, C, G and T) into a
growing copy nucleic acid. For example, the recombinant polymerase
optionally displays a specific activity for a natural nucleotide
that is at least about 5% as high (e.g., 5%, 10%, 25%, 50%, 75%,
100% or higher), as a corresponding wild-type polymerase and a
processivity with natural nucleotides in the presence of a template
that is at least 5% as high (e.g., 5%, 10%, 25%, 50%, 75%, 100% or
higher) as the wild-type polymerase in the presence of the natural
nucleotide. Optionally, the recombinant polymerase displays a
k.sub.cat/K.sub.m or V.sub.max/K.sub.m for a naturally occurring
nucleotide that is at least about 5% as high (e.g., about 5%, 10%,
25%, 50%, 75% or 100% or higher) as the wild-type polymerase.
[0021] The nucleotide analogue and a DNA template are optionally
included in compositions of the invention, e.g., in which the
recombinant polymerase incorporates the nucleotide analogue into a
copy nucleic acid in response to the template DNA. The template DNA
can be linear or circular DNA, and in certain sequencing
applications is desirable a circular template. Thus, the
composition can be present in a DNA amplification and/or sequencing
system. Optionally, in one class of embodiments, the sequencing
system comprises a Zero Mode Waveguide.
[0022] Methods of making and using the compositions are also
features of the invention. For example, in one aspect, methods of
making a DNA e.g., comprising one or more nucleotide analogue
residues are provided. In these methods, a reaction mixture is
provided. The reaction mixture typically includes those components
that can at least partially replicate a template, e.g., a template,
nucleotides, the polymerase and a replication initiating moiety
that complexes with the template, or is integral to it, to prime
the polymerase. The replication initiating moiety in this context
is any moiety that can serve as a site to initiate the polymerase,
e.g., a separate oligonucleotide complementary to the template, a
hairpin or other self-complementary region of a template (e.g., a
hairpin in a single-stranded template), a terminal protein, or the
like. The polymerase is a recombinant polymerase capable of at
least partially replicating the template in a template-dependent
polymerase extension reaction (e.g., using the replication
initiation moiety as a site of initiation). Typically, the one or
more nucleotides comprise a nucleotide analogue. In preferred
aspects, at least one, preferably two or more, three or more or at
least four nucleotides are nucleotide analogues. The recombinant
DNA polymerase has a modified active site (a region of the
polymerase that, when modified, results in an alteration in an
activity of the polymerase) that is homologous to a wild-type
active site of a wild-type DNA polymerase. As discussed in the
context of the compositions above, the modified active site can
include one or more structural modification relative to the wild
type active site that improves the activity of the enzyme toward
one or more natural nucleotides and/or nucleotide analogues. In at
least one example, and without being bound to any particular theory
of operation, the modification to the active site reduces steric
inhibition for entry of the nucleotide analogue into the modified
active site and/or the modification is complementary with one or
more non-natural features of the nucleotide analogue.
[0023] The mixture is reacted such that the recombinant polymerase
replicates at least a portion of the template in a
template-dependent manner, whereby at least one nucleotide analogue
residue is incorporated into the resulting DNA. Incorporation of
the analogue can result in the incorporation of a non-standard
residue into the extended DNA (e.g., as a labeled nucleotide
residue), or action of the polymerase can modify the analogue such
that the nucleotide analogue residue incorporated into the extended
DNA is structurally the same as a standard nucleotide residue. For
example, in the latter embodiment, a variety of labels are cleaved
by action of the polymerase, e.g., certain phosphate labels
discussed in more detail herein are cleaved from the nucleotide
analogue as it is incorporated into the growing DNA (typically
providing a signal upon release of the label).
[0024] In a related class of methods, a reaction mixture is
provided that includes a template, a replication initiating moiety,
a template-dependent recombinant polymerase and one or more
nucleotides. The one or more nucleotides include a phosphate
labeled nucleotide. A K.sub.m value of the recombinant polymerase
for the nucleotide analogue is lower than a K.sub.m for a
corresponding homologous wild-type polymerase for the nucleotide
analogue. The mixture is reacted such that the polymerase at least
partially replicates the template in a template-dependent manner,
e.g., whereby at least one nucleotide analogue residue is
incorporated into the resulting DNA. As noted previously, once
incorporated, the residue can be the same as a natural nucleotide
residue, or can be different from a natural nucleotide residue.
[0025] In another related class of methods of making a DNA, a
reaction mixture that includes a template, a replication initiating
moiety that complexes with or is integral to the template, a
polymerase capable of replicating at least a portion of the
template using the moiety in a template-dependent polymerase
extension reaction, and one or more nucleotide is provided. Here
again, the one or more nucleotide typically includes a labeled
phosphate nucleotide analogue. The polymerase in this class of
embodiments is homologous to a .PHI.29 DNA polymerase. The
polymerase has a K.sub.m for 488dC4P, A568dC4P, or both, that is
less than about 75% of a K.sub.m of a GST-N62D .PHI.29 DNA
polymerase for 488dC4P, A568dC4P or both. For example, the K.sub.m
for 488dC4P, A568dC4P can be about 40% or less than GST-N62D
.PHI.29 DNA polymerase, or, e.g., about 15% or less. The mixture is
reacted such that the polymerase replicates at least a portion of
the template.
[0026] The polymerases used in the methods can be any of those
noted above with reference to the compositions. The properties of
the polymerases used in the methods can be any of those noted in
reference to compositions. For example, the polymerase optionally
has a k.sub.cat/K.sub.m for the nucleotide analogue that is higher
than a k.sub.cat/K.sub.m of a wild-type .PHI.29 for the nucleotide
analogue. Similarly, the nucleotide analogues used in the methods
can be any of those noted in reference to the compositions herein.
The recombinant polymerases herein can have a K.sub.m for the
nucleotide analogue that is e.g., about 90% as high, about 80% as
high, about 75% as high, about 60% as high, about 50% as high,
about 40% as high, about 25% as high, about 15% as high, about 10%
as high, or less than about 5% as high as a K.sub.m of a naturally
occurring polymerase homologous to the recombinant polymerase. The
recombinant polymerase optionally has an increased rate of binding
of the nucleotide analogue, an increased rate of product release,
and/or a decreased branching rate, as compared to the corresponding
wild-type polymerase.
[0027] In addition to methods of using the compositions herein, the
present invention also includes methods of making the compositions.
For example, in one aspect, a method of making a recombinant DNA
polymerase (e.g., any of those discussed with respect to the
compositions herein) is provided. For example, the methods can
include structurally modeling a first polymerase, e.g., using any
available crystal structure and molecular modeling software or
system. Based on the modeling, one or more steric inhibition
feature or complementarity feature affecting nucleotide access to
the active site and/or binding of a nucleotide analogue within the
active site region is identified, e.g., in the active site or
proximal to it. The first DNA polymerase is mutated to reduce or
remove at least one steric inhibition feature or to add the
complementarity feature.
[0028] The method can additionally include screening or other
protocols to determine whether the resulting recombinant polymerase
displays a modified activity for a nucleotide analogue as compared
to the first DNA polymerase. For example, k.sub.cat, K.sub.m,
V.sub.max, or k.sub.cat/K.sub.m of the recombinant DNA polymerase
for the nucleotide analogue can be determined. Further, k.sub.cat,
K.sub.m, V.sub.max, or k.sub.cat/K.sub.m of the recombinant DNA
polymerase for a natural nucleotide can also be determined (e.g.,
where the polymerase desirably includes both analogue and natural
nucleotide incorporation activity).
[0029] A library of recombinant DNA polymerases can be made and
screened for these properties. For example, a plurality of members
of the library can be made to include one or more steric inhibition
feature mutation and/or a mutation to produce complementary with
one or more non-natural features of the nucleotide analogue, that
is then screened for the properties of interest. In general, the
library can be screened to identify at least one member comprising
a modified activity of interest.
[0030] In an additional aspect, the invention includes
computer-implemented methods, e.g., for modeling enzyme kinetics.
The methods include, e.g., defining a plurality of polymerase state
transitions for discrete time steps during a template-based
polymerization reaction; defining a plurality of rate transition
rates between the states; generating a multidimensional probability
matrix of possible states, based upon a given nucleic acid template
sequence, nucleotides in a reaction mixture and the polymerase
state transitions; and, storing the multidimensional probability
matrix in a computer readable medium.
[0031] A variety of features of the method can vary. For example,
the polymerase state transitions are optionally user-selectable.
The rate transition rates between the states optionally vary
depending on nucleotide concentration, template sequence and
position of the polymerase along the template. The nucleotides in
the reaction mixture optionally comprise one or more nucleotide
analogues. The rate transition rates between states optionally
include a conformational transition rate for the polymerase during
use of the nucleotide analogues by the polymerase, with the rate
set to be equal to a conformational transition rate for a natural
nucleotide. The multidimensional probability matrix is optionally
automatically generated based upon the template sequence, a
standardized matrix of probability states, and the nucleotides in
the reaction mixture. The probability matrix is optionally
simplified by assuming that all possible Watson-Crick base pairings
are equivalent in all state transitions.
[0032] Similarly, a second reagent concentration matrix is
optionally generated to account for reagent concentration changes
that result from position of the polymerase along a template, based
on an output of the probability matrix. The probability matrix is
optionally vectorized for multiple templates and the resulting
vectorized probability matrix can be multiplied by the
multidimensional probability matrix to provide a state distribution
matrix. An exponential time factor for the probability matrix can
be used to account for repeated sequences within the template
sequence. A polymerase nucleotide mismatch fraction using either a
continuum model or a counting model can be defined.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0034] FIG. 1 schematically depicts a vector for expression of
tagged N62D Phi 29 DNA polymerase.
[0035] FIG. 2 Panel A presents a sequence alignment for Phi 29-like
polymerases in the region surrounding residues 505-525 (Phi29 SEQ
ID NO:35, B103 SEQ ID NO:36, PZA SEQ ID NO:37, M2 SEQ ID NO:38, G1
SEQ ID NO:39, cp-1 SEQ ID NO:40). Panel B illustrates the structure
of Phi 29 with (top) and without (bottom) residues 505-525. Views
of the structures from three different angles are shown.
[0036] FIG. 3 Panel A presents a sequence alignment for Phi 29-like
polymerases in the region surrounding E375 of Phi 29 (Phi29 SEQ ID
NO:41, B103 SEQ ID NO:42, PZA SEQ ID NO:43, M2 SEQ ID NO:44, G1 SEQ
ID NO:45, cp-1 SEQ ID NO:46). Panels B illustrates the structure of
Phi 29 (top) and an E375H mutant (bottom). Views of the structures
from three different angles are shown.
[0037] FIG. 4 Panel A presents a sequence alignment for Phi 29-like
polymerases in the region surrounding E486 of Phi 29 (Phi29 SEQ ID
NO:47, B103 SEQ ID NO:48, PZA SEQ ID NO:49, M2 SEQ ID NO:50, G1 SEQ
ID NO:51, cp-1 SEQ ID NO:52). Panels B illustrates the structure of
Phi 29 (top) and an E486A mutant (bottom). Views of the structures
from three different angles are shown.
[0038] FIG. 5 Panel A shows a sequence alignment for Phi 29-like
polymerases in the region surrounding K512 of Phi 29 (Phi29 SEQ ID
NO:53, B103 SEQ ID NO:54, PZA SEQ ID NO:55, M2 SEQ ID NO:56, G1 SEQ
ID NO:57, cp-1 SEQ ID NO:58). Panels B illustrates the structure of
Phi 29 (top) and a K512A mutant (bottom). Views of the structures
from three different angles are shown.
[0039] FIG. 6 Panel A shows a sequence alignment for Phi 29-like
polymerases in the region surrounding K135 of Phi 29 (Phi29 SEQ ID
NO:59, B103 SEQ ID NO:60, PZA SEQ ID NO:61, M2 SEQ ID NO:62, G1 SEQ
ID NO:63, cp-1 SEQ ID NO:64). Panels B illustrates the structure of
Phi 29 (top) and a K135A mutant (bottom). Views of the structures
from three different angles are shown.
[0040] FIG. 7 Panel A schematically illustrates a FRET stopped flow
assay used to determine rates of binding and product release.
Results of the assay are shown in Panels B-D, for Phi29 N62D (Panel
B), N62D:E375Y (Panel C), and N62D:E375W (Panel D).
[0041] FIG. 8 Panel A schematically illustrates a FRET stopped flow
assay used to determine branching rate. Results of the assay are
shown in Panels B-D, for Phi29 N62D (Panel B), N62D:E375Y (Panel
C), and N62D:E375W (Panel D).
[0042] FIG. 9 depicts a plot of kinetic matrix jump size vs.
concentration drop.
DETAILED DISCUSSION OF THE INVENTION
Overview
[0043] A variety of technologies rely on the incorporation of
labels into nucleic acids to observe the results of an experiment.
For example, the outcome of sequencing, nucleic acid amplification
and nick translation reactions are all typically monitored by
labeling product nucleic acids. This is often done by covalently or
non-covalently binding labels to the product nucleic acids, e.g.,
by binding labeled probes to the product nucleic acid. In other
approaches, nucleotide analogues are incorporated into product
nucleic acids during synthesis of the product nucleic acid. This
typically occurs, e.g., in sequencing by incorporation methods, and
certain real-time PCR (RT-PCR) and real-time LCR reactions
(RT-LCR). A label present on the analogue can be incorporated into
the DNA, or it can be released by action of the polymerase.
Incorporation or release of the label can be monitored to monitor
incorporation of an analogue residue into the product nucleic
acid.
[0044] The present invention provides new polymerases that
incorporate nucleotide analogues, such as dye labeled phosphate
labeled analogues, into a growing template copy, during DNA
amplification. These polymerases are modified such that the active
site of the polymerase is modified to reduce steric entry
inhibition of the analogue into the active site (facilitating entry
of the nucleotide analogue into the active site) and/or to provide
complementarity with one or more non-natural features of the
nucleotide analogue.
[0045] These new polymerases are particularly well-suited to DNA
amplification (e.g., RT-PCR and RT-LCR) and/or sequencing
applications, e.g., in the context of amplification or sequencing
protocols that include incorporation of labeled analogues into DNA
amplicons.
DNA Polymerases
[0046] DNA polymerases that can be modified to interact with
nucleotide analogues by reducing steric entry inhibition into the
active site, or by adding features complementary to the analogues,
are generally available. DNA polymerases have relatively recently
been classified into six main groups based upon various
phylogenetic relationships, e.g., with E. coli Pol I (class A), E.
coli Pol II (class B), E. coli Pol III (class C), Euryarchaeotic
Pol II (class D), human Pol beta (class X), and E. coli UmuC/DinB
and eukaryotic RAD30/xeroderma pigmentosum variant (class Y). For a
review of recent nomenclature, see, e.g., Burgers et al. (2001)
"Eukaryotic DNA polymerases: proposal for a revised nomenclature" J
Biol Chem. 276(47):43487-90. For a review of polymerases, see,
e.g., Hubscher et al. (2002) EUKARYOTIC DNA POLYMERASES Annual
Review of Biochemistry Vol. 71: 133-163; Alba (2001) "Protein
Family Review: Replicative DNA Polymerases" Genome Biology
2(1):reviews 3002.1-3002.4; and Steitz (1999) "DNA polymerases:
structural diversity and common mechanisms" J Biol Chem
274:17395-17398. The basic mechanisms of action for many
polymerases have been determined. The sequences of literally
hundreds of polymerases are publicly available, and the crystal
structures for many of these have been determined, or can be
inferred based upon similarity to solved crystal structures for
homologous polymerases. For example, the crystal structure of
.PHI.29 is available.
[0047] Available DNA polymerase enzymes have also been modified in
any of a variety of ways, e.g., to reduce or eliminate exonuclease
activities (many native DNA polymerases have a proof-reading
exonuclease function that interferes with, e.g., sequencing
applications), to simplify production by making protease digested
enzyme fragments such as the Klenow fragment recombinant, etc. Any
of these available polymerases can be modified in accordance with
the invention to reduce steric inhibition to analogue entry into
the active site, or to provide features complementary to the
analogue. Many such polymerases that are suitable for modification
are available, e.g., for use in sequencing, labeling and
amplification technologies. For example, Human DNA Polymerase Beta
is available from R&D systems. DNA polymerase I is available
from Epicenter, GE Health Care, Invitrogen, New England Biolabs,
Promega, Roche Applied Science, Sigma Aldrich and many others. The
Klenow fragment of DNA Polymerase I is available in both
recombinant and protease digested versions, from, e.g., Ambion,
Chimerx, eEnzyme LLC, GE Health Care, Invitrogen, New England
Biolabs, Promega, Roche Applied Science, Sigma Aldrich and many
others. .PHI.29 DNA polymerase is available from e.g., Epicenter.
Poly A polymerase, reverse transcriptase, Sequenase, SP6 DNA
polymerase, T4 DNA polymerase, T7 DNA polymerase, and a variety of
thermostable DNA polymerases (Taq, hot start, titanium Taq, etc.)
are available from a variety of these and other sources. Recent
commercial DNA polymerases include Phusion.TM. High-Fidelity DNA
Polymerase is available from New England Biolabs; GoTaq.RTM. Flexi
DNA Polymerase available from Promega; RepliPHI.TM. .PHI.29 DNA
Polymerase from EPICENTRE; PfuUltra.TM. Hotstart DNA Polymerase
available from Stratagene; KOD HiFi DNA Polymerase is available
from Novagen and many others. Biocompare(dot)com provides
comparisons of many different commercially available
polymerases.
[0048] DNA polymerases that are preferred substrates for mutation
to reduce steric inhibition or to incorporate features
complementary to the nucleotide analogue include Taq polymerases,
exonuclease deficient Taq polymerases, E. coli DNA Polymerase 1,
Klenow fragment, reverse transcriptases, .PHI.29 related
polymerases including wild type .PHI.29 polymerase derivatives of
such polymerases such as exonuclease deficient forms, T7 DNA
Polymerase, T5 DNA Polymerase, an RB69 polymerase, etc. For
example, the recombinant DNA polymerase can be homologous to a
wild-type or exonuclease deficient .PHI.29 DNA polymerase, e.g., as
described in U.S. Pat. No. 5,001,050, 5,198,543, or 5,576,204.
Similarly, the recombinant DNA polymerase can be homologous to
.PHI.29, B103, GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1,
PZF, SFS, Cp-5, Cp-7, PR4, PR5, PR722, or L17, or the like.
Nucleotide Analogues
[0049] As discussed, various polymerases of the invention can
incorporate one or more nucleotide analogues into a growing
oligonucleotide chain. Upon incorporation, the analogue can leave a
residue that is the same or different than a natural nucleotide in
the growing oligonucleotide (the polymerase can incorporate any
non-standard moiety of the analogue, or can cleave it off during
incorporation into the oligonucleotide). A "nucleotide analogue"
herein is a compound, that, in a particular application, functions
in a manner similar or analogous to a naturally occurring
nucleoside triphosphate (a "nucleotide"), and does not otherwise
denote any particular structure. A nucleotide analogue is an
analogue other than a standard naturally occurring nucleotide,
i.e., other than A, G, C, T, or U, though upon incorporation into
the oligonucleotide, the resulting residue in the oligonucleotide
can be the same as (or different from) an A, G, C, T or U
residue.
[0050] Many nucleotide analogues are available. These include
analogue structures with core similarity to naturally occurring
nucleotides, such as those that comprise one or more substituent on
a phosphate, sugar or base moiety of the nucleoside or nucleotide
relative to a naturally occurring nucleoside or nucleotide. In one
embodiment, a nucleotide analogue can include one or more extra
phosphate containing groups, relative to a nucleoside triphosphate.
For example, a variety of nucleotide analogues that comprise, e.g.,
from 4-6 phosphates are described in detail in U.S. patent
application Ser. No. 11/241,809, filed Sep. 29, 2005, and
incorporated herein by reference in its entirety for all
purposes.
[0051] For example, the analogue can include a labeled compound of
the formula:
##STR00003##
wherein B is a nucleobase (and optionally includes a label); S is
selected from a sugar moiety, an acyclic moiety or a carbocyclic
moiety (and optionally includes a label); L is an optional
detectable label; R.sub.1 is selected from O and S; R.sub.2,
R.sub.3 and R.sub.4 are independently selected from O, NH, S,
methylene, substituted methylene, C(O), C(CH.sub.2), CNH.sub.2,
CH.sub.2CH.sub.2, C(OH)CH.sub.2R where R is 4-pyridine or
1-imidazole, provided that R.sub.4 may additionally be selected
from
##STR00004##
R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.11 and R.sub.13 are, when
present, each independently selected from O, BH.sub.3, and S; and
R.sub.9, R.sub.10 and R.sub.12 are independently selected from O,
NH, S, methylene, substituted methylene, CNH.sub.2,
CH.sub.2CH.sub.2, C(OH)CH.sub.2R where R is 4-pyridine or
1-imidazole. In some cases, phosphonate analogs may be employed as
the analogs, e.g., where one of R.sub.2, R.sub.3, R.sub.4, R.sub.9,
R.sub.10 or R.sub.12 are not O, e.g., they are methyl etc. See,
e.g., U.S. patent application Ser. No. 11/241,809, previously
incorporated herein by reference in its entirety for all
purposes.
[0052] The base moiety incorporated into the analogue is generally
selected from any of the natural or non-natural nucleobases or
nucleobase analogs, including, e.g., purine or pyrimidine bases
that are routinely found in nucleic acids and available nucleic
acid analogs, including adenine, thymine, guanine, cytidine,
uracil, and in some cases, inosine. As noted, the base optionally
includes a label moiety. For convenience, nucleotides and
nucleotide analogs are generally referred to based upon their
relative analogy to naturally occurring nucleotides. As such, an
analogue that operates, functionally, like adenosine triphosphate,
may be generally referred to herein by the shorthand letter A.
Likewise, the standard abbreviations of T, G, C, U and I, may be
used in referring to analogs of naturally occurring nucleosides and
nucleotides typically abbreviated in the same fashion. In some
cases, a base may function in a more universal fashion, e.g.,
functioning like any of the purine bases in being able to hybridize
with any pyrimidine base, or vice versa. The base moieties used in
the present invention may include the conventional bases described
herein or they may include such bases substituted at one or more
side groups, or other fluorescent bases or base analogs, such as
1,N6 ethenoadenosine or pyrrolo C, in which an additional ring
structure renders the B group neither a purine nor a pyrimidine.
For example, in certain cases, it may be desirable to substitute
one or more side groups of the base moiety with a labeling group or
a component of a labeling group, such as one of a donor or acceptor
fluorophore, or other labeling group. Examples of labeled
nucleobases and processes for labeling such groups are described
in, e.g., U.S. Pat. Nos. 5,328,824 and 5,476,928, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0053] In the analogues, the S group is optionally a sugar moiety
that provides a suitable backbone for a synthesizing nucleic acid
strand. For example, the sugar moiety is optionally selected from a
D-ribosyl, 2' or 3' D-deoxyribosyl, 2',3'-D-dideoxyribosyl,
2',3'-D-didehydrodideoxyribosyl, 2' or 3' alkoxyribosyl, 2' or 3' a
minoribosyl, 2' or 3' mercaptoribosyl, 2' or 3' alkothioribosyl,
acyclic, carbocyclic or other modified sugar moieties. A variety of
carbocyclic or acyclic moieties can be incorporated as the "S"
group in place of a sugar moiety, including, e.g., those described
in published U.S. Patent Application No. 2003/0124576, previously
incorporated herein by reference in its entirety for all
purposes.
[0054] For most cases, the phosphorus containing chain in the
analogues, e.g., a triphosphate in conventional NTPs, is preferably
coupled to the 5' hydroxyl group, as in natural nucleoside
triphosphates. However, in some cases, the phosphorus containing
chain is linked to the S group by the 3' hydroxyl group.
[0055] L generally refers to a detectable labeling group that is
coupled to the terminal phosphorus atom via the R.sub.4 (or
R.sub.10 or R.sub.12) group. The labeling groups employed in the
analogs of the invention may comprise any of a variety of
detectable labels. Detectable labels generally denote a chemical
moiety that provides a basis for detection of the analogue compound
separate and apart from the same compound lacking such a labeling
group. Examples of labels include, e.g., optical labels, e.g.,
labels that impart a detectable optical property to the analogue,
electrochemical labels, e.g., labels that impart a detectable
electrical or electrochemical property to the analogue, physical
labels, e.g., labels that impart a different physical or spatial
property to the analogue, e.g., a mass tag or molecular volume tag.
In some cases individual labels or combinations may be used that
impart more than one of the aforementioned properties to the
analogs of the invention.
[0056] Optionally, the labeling groups incorporated into the
analogs comprise optically detectable moieties, such as
luminescent, chemiluminescent, fluorescent, fluorogenic,
chromophoric and/or chromogenic moieties, with fluorescent and/or
fluorogenic labels being preferred. A variety of different label
moieties are readily employed in nucleotide analogs. Such groups
include fluorescein labels, rhodamine labels, cyanine labels (i.e.,
Cy3, Cy5, and the like, generally available from the Amersham
Biosciences division of GE Healthcare), the Alexa family of
fluorescent dyes and other fluorescent and fluorogenic dyes
available from Molecular Probes/Invitrogen, Inc., and described in
`The Handbook--A Guide to Fluorescent Probes and Labeling
Technologies, Tenth Edition` (2005) (available from Invitrogen,
Inc./Molecular Probes). A variety of other fluorescent and
fluorogenic labels for use with nucleoside polyphosphates, and
which would be applicable to the nucleotide analogues incorporated
by the polymerases of the present invention are described in, e.g.,
Published U.S. Patent Application No. 2003/0124576, the full
disclosure of which is incorporated herein in its entirety for all
purposes.
[0057] Additional details regarding analogues and methods of making
such analogues can be found in U.S. patent application Ser. No.
11/241,809, filed Sep. 29, 2005, and incorporated herein by
reference in its entirety for all purposes.
[0058] Thus, in one illustrative example, the analogue can be a
phosphate analogue (e.g., an analogue that has more than the
typical number of phosphates found in nucleoside triphosphates)
that include, e.g., an Alexa dye label. For example, an Alexa488
dye can be labeled on a deltaphosphate (denoted, e.g., A488dC4P),
or an Alexa568 or Alexa633 dye can be used (e.g., A568dC4P, and
A633dC4P respectively), or an Alexa546 dye can be used (e.g.,
A546dG4P), or an Alexa594 dye can be used (e.g., A594dT4P).
Similarly, to facilitate color separation, a pair of fluorophores
exhibiting FRET (fluorescence resonance energy transfer) can be
labeled on a delta phosphate of a tetraphosphate analog (denoted,
e.g., FAM-amb-A532dG4P or FAM-amb-A594dT4P).
Modifying DNA Polymerases to Reduce Steric Hindrance Features
and/or to Add Complementarity Features
[0059] Structure-Based Design of Recombinant Polymerases
[0060] Structural data for a polymerase can be used to conveniently
identify amino acid residues as candidates for mutagenesis to
create recombinant polymerases having modified active site regions.
For example, analysis of the three-dimensional structure of a
polymerase can identify residues that sterically hinder access to
the active site by a natural nucleotide or nucleotide analogue or
analogue thereof or that can be mutated to introduce a feature
complementary to a non-natural feature of the analogue, e.g., by
adding or altering charge, hydrophobicity, size, or the like.
[0061] The three-dimensional structures of a large number of DNA
polymerases have been determined by x-ray crystallography and
nuclear magnetic resonance (NMR) spectroscopy, including the
structures of polymerases with bound templates, nucleotides, and/or
nucleotide analogues. Many such structures are freely available for
download from the Protein Data Bank, at (www(dot)rcsb(dot)org/pdb.
Structures, along with domain and homology information, are also
freely available for search and download from the National Center
for Biotechnology Information's Molecular Modeling DataBase, at
www(dot)ncbi(dot)nlm(dot)nih(dot)gov/Structure/MMDB/mmdb(dot)shtml.
The structures of additional polymerases can be modeled, for
example, based on homology of the polymerases with polymerases
whose structures have already been determined. Alternatively, the
structure of a given polymerase, optionally complexed with a
nucleotide analogue, or the like, can be determined.
[0062] Techniques for crystal structure determination are well
known. See, for example, McPherson (1999) Crystallization of
Biological Macromolecules Cold Spring Harbor Laboratory; Bergfors
(1999) Protein Crystallization International University Line;
Mullin (1993) Crystallization Butterworth-Heinemann; Stout and
Jensen (1989) X-ray structure determination: a practical guide, 2nd
Edition Wiley Publishers, New York; Ladd and Palmer (1993)
Structure determination by X-ray crystallography, 3rd Edition
Plenum Press, New York; Blundell and Johnson (1976) Protein
Crystallography Academic Press, New York; Glusker and Trueblood
(1985) Crystal structure analysis: A primer, 2nd Ed. Oxford
University Press, New York; International Tables for
Crystallography, Vol. F. Crystallography of Biological
Macromolecules; McPherson (2002) Introduction to Macromolecular
Crystallography Wiley-Liss; McRee and David (1999) Practical
Protein Crystallography, Second Edition Academic Press; Drenth
(1999) Principles of Protein X-Ray Crystallography (Springer
Advanced Texts in Chemistry) Springer-Verlag; Fanchon and
Hendrickson (1991) Chapter 15 of Crystallographic Computing, Volume
5 IUCr/Oxford University Press; Murthy (1996) Chapter 5 of
Crystallographic Methods and Protocols Humana Press; Dauter et al.
(2000) "Novel approach to phasing proteins: derivatization by short
cryo-soaking with halides" Acta Cryst. D56:232-237; Dauter (2002)
"New approaches to high-throughput phasing" Curr. Opin. Structural
Biol. 12:674-678; Chen et al. (1991) "Crystal structure of a bovine
neurophysin-II dipeptide complex at 2.8 .ANG. determined from the
single-wavelength anomalous scattering signal of an incorporated
iodine atom" Proc. Natl. Acad. Sci. USA, 88:4240-4244; and Gavira
et al. (2002) "Ab initio crystallographic structure determination
of insulin from protein to electron density without crystal
handling" Acta Cryst. D58:1147-1154.
[0063] In addition, a variety of programs to facilitate data
collection, phase determination, model building and refinement, and
the like are publicly available. Examples include, but are not
limited to, the HKL2000 package (Otwinowski and Minor (1997)
"Processing of X-ray Diffraction Data Collected in Oscillation
Mode" Methods in Enzymology 276:307-326), the CCP4 package
(Collaborative Computational Project (1994) "The CCP4 suite:
programs for protein crystallography" Acta Crystallogr D
50:760-763), SOLVE and RESOLVE (Terwilliger and Berendzen (1999)
Acta Crystallogr D 55 (Pt 4):849-861), SHELXS and SHELXD (Schneider
and Sheldrick (2002) "Substructure solution with SHELXD" Acta
Crystallogr D Biol Crystallogr 58:1772-1779), Refmac5 (Murshudov et
al. (1997) "Refinement of Macromolecular Structures by the
Maximum-Likelihood Method" Acta Crystallogr D 53:240-255), PRODRG
(van Aalten et al. (1996) "PRODRG, a program for generating
molecular topologies and unique molecular descriptors from
coordinates of small molecules" J Comput Aided Mol Des 10:255-262),
and O (Jones et al. (1991) "Improved methods for building protein
models in electron density maps and the location of errors in these
models" Acta Crystallogr A 47 (Pt 2):110-119).
[0064] Techniques for structure determination by NMR spectroscopy
are similarly well described in the literature. See, e.g., Cavanagh
et al. (1995) Protein NMR Spectroscopy: Principles and Practice,
Academic Press; Levitt (2001) Spin Dynamics: Basics of Nuclear
Magnetic Resonance, John Wiley & Sons; Evans (1995)
Biomolecular NMR Spectroscopy, Oxford University Press; Wuthrich
(1986) NMR of Proteins and Nucleic Acids (Baker Lecture Series),
Kurt Wiley-Interscience; Neuhaus and Williamson (2000) The Nuclear
Overhauser Effect in Structural and Conformational Analysis, 2nd
Edition, Wiley-VCH; Macomber (1998) A Complete Introduction to
Modern NMR Spectroscopy, Wiley-Interscience; Downing (2004) Protein
NMR Techniques (Methods in Molecular Biology), 2nd edition, Humana
Press; Clore and Gronenborn (1994) NMR of Proteins (Topics in
Molecular and Structural Biology), CRC Press; Reid (1997) Protein
NMR Techniques, Humana Press; Krishna and Berliner (2003) Protein
NMR for the Millenium (Biological Magnetic Resonance), Kluwer
Academic Publishers; Kiihne and De Groot (2001) Perspectives on
Solid State NMR in Biology (Focus on Structural Biology, 1), Kluwer
Academic Publishers; Jones et al. (1993) Spectroscopic Methods and
Analyses: NMR, Mass Spectrometry, and Related Techniques (Methods
in Molecular Biology, Vol. 17), Humana Press; Goto and Kay (2000)
Curr. Opin. Struct. Biol. 10:585; Gardner (1998) Annu. Rev.
Biophys. Biomol. Struct. 27:357; Wiithrich (2003) Angew. Chem. Int.
Ed. 42:3340; Bax (1994) Curr. Opin. Struct. Biol. 4:738; Pervushin
et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12366; Fiaux et al.
(2002) Nature 418:207; Fernandez and Wider (2003) Curr. Opin.
Struct. Biol. 13:570; Ellman et al. (1992) J. Am. Chem. Soc.
114:7959; Wider (2000) BioTechniques 29:1278-1294; Pellecchia et
al. (2002) Nature Rev. Drug Discov. (2002) 1:211-219; Arora and
Tamm (2001) Curr. Opin. Struct. Biol. 11:540-547; Flaux et al.
(2002) Nature 418:207-211; Pellecchia et al. (2001) J. Am. Chem.
Soc. 123:4633-4634; and Pervushin et al. (1997) Proc. Natl. Acad.
Sci. USA 94:12366-12371.
[0065] The structure of a polymerase with a given nucleotide
analogue incorporated into the active site can, as noted, be
directly determined, e.g., by x-ray crystallography or NMR
spectroscopy, or the structure can be modeled based on the
structure of the polymerase and/or a structure of a polymerase with
a natural nucleotide bound. The active site region of the
polymerase can be identified, for example, by homology with other
polymerases, examination of polymerase-template or
polymerase-nucleotide co-complexes, biochemical analysis of mutant
polymerases, and/or the like. The position of a nucleotide analogue
in the active site can be modeled, for example, by projecting the
location of non-natural features of the analogue (e.g., additional
phosphate or phosphonate groups in the phosphorus containing chain
linked to the nucleotide, e.g., tetra, penta or hexa phosphate
groups, detectable labeling groups, e.g., fluorescent dyes, or the
like) based on the previously determined location of another
nucleotide or nucleotide analogue in the active site.
[0066] Such modeling of the nucleotide analogue in the active site
can involve simple visual inspection of a model of the polymerase,
for example, using molecular graphics software such as the PyMOL
viewer (open source, freely available on the World Wide Web at
www(dot)pymol(dot)org) or Insight II (commercially available from
Accelrys at (www(dot)accelrys(dot)com/products/insight).
Alternatively, modeling of the nucleotide analogue in the active
site of the polymerase or a putative mutant polymerase, for
example, can involve computer-assisted docking, molecular dynamics,
free energy minimization, and/or like calculations. Such modeling
techniques have been well described in the literature; see, e.g.,
Babine and Abdel-Meguid (eds.) (2004) Protein Crystallography in
Drug Design, Wiley-VCH, Weinheim; Lyne (2002) "Structure-based
virtual screening: An overview" Drug Discov. Today 7:1047-1055;
Molecular Modeling for Beginners, at
(www(dot)usm(dot)maine(dot)edu/.about.rhodes/SPVTut/index(dot)html;
and Methods for Protein Simulations and Drug Design at
(www(dot)dddc(dot)ac(dot)cn/embo04; and references therein.
Software to facilitate such modeling is widely available, for
example, the CHARMm simulation package, available academically from
Harvard University or commercially from Accelrys (at
www(dot)accelrys(dot)com), the Discover simulation package
(included in Insight II, supra), and Dynama (available at
(www(dot)cs(dot)gsu(dot)edu/.about.cscrwh/progs/progs(dot)html).
See also an extensive list of modeling software at
(www(dot)netsci(dot)org/Resources/Software/Modeling/MMMD/top(dot)html.
[0067] Visual inspection and/or computational analysis of a
polymerase model can identify relevant features of the active site
region, including, for example, residues that can sterically
inhibit entry of a nucleotide analogue into the active site (e.g.,
residues undesirably close to the projected location of one or more
atoms within the analogue when the analogue is bound to the
polymerase). Such a residue can, for example, be deleted or
replaced with a residue having a smaller side chain; for example,
many residues can be conveniently replaced with a residue having
similar characteristics but a shorter amino acid side chain, or,
e.g., with alanine. Similarly, residues that can be altered to
introduce desirable interactions with the nucleotide analogue can
be identified. Such a residue can be replaced with a residue that
is complementary with a non-natural feature of the analogue, for
example, with a residue that can hydrogen bond to the analogue
(e.g., serine, threonine, histidine, asparagine, or glutamine), a
hydrophobic residue that can interact with a hydrophobic group on
the analogue, an aromatic residue that can provide favorable
hydrophobic interactions with a group on the analogue (e.g., a
fluorophore), an aromatic residue that can engage in a .pi.-.pi. or
edge-face stacking interaction with an aromatic group in the
analogue, a residue that can engage in a cation-.pi. interaction
with the analogue, or a charged residue (e.g., aspartic or glutamic
acid, or lysine, arginine, or histidine) that can electrostatically
interact with an oppositely charged moiety on the analogue (e.g.,
an additional phosphate group).
[0068] As just one specific example of such structure-based design,
inspection of a model of the .PHI.29 polymerase identified the
.DELTA.505-525 domain and residues K135, E486, and K512 as
potentially sterically inhibiting entry of an analogue into the
active site, and suggested that mutation of E375 to histidine,
lysine, or arginine would introduce a positive charge complementary
to a non-natural tetra phosphate on the analogue. Similarly,
inspection of the model suggested that mutation of E375 to an
aromatic residue such as tryptophan, tyrosine, or phenylalanine
would improve hydrophobic interactions with a fluorophore on the
analogue. See Examples 2 and 3 below for additional details.
[0069] Thus, in addition to methods of using the polymerases and
other compositions herein, the present invention also includes
methods of making the polymerases. As described, methods of making
a recombinant DNA polymerase can include structurally modeling a
first polymerase, e.g., using any available crystal structure and
molecular modeling software or system. Based on the modeling, one
or more steric inhibition feature or complementarity feature
affecting nucleotide access to the active site and/or binding of a
nucleotide analogue within the active site region is identified,
e.g., in the active site or proximal to it. The first DNA
polymerase is mutated to reduce or remove at least one steric
inhibition feature or to add the complementarity feature.
[0070] Mutating Active Site Regions
[0071] Various types of mutagenesis are optionally used in the
present invention, e.g., to modify polymerases to produce variants
comprising complementarity features and or to reduce steric
hindrance features, e.g., in accordance with polymerase models and
model predictions as discussed above. In general, any available
mutagenesis procedure can be used for making such mutants. Such
mutagenesis procedures optionally include selection of mutant
nucleic acids and polypeptides for one or more activity of interest
(e.g., improved K.sub.m, V.sub.max, k.sub.cat etc., for a
nucleotide analogue). Procedures that can be used include, but are
not limited to: site-directed point mutagenesis, random point
mutagenesis, in vitro or in vivo homologous recombination (DNA
shuffling), mutagenesis using uracil containing templates,
oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA
mutagenesis, mutagenesis using gapped duplex DNA, point mismatch
repair, mutagenesis using repair-deficient host strains,
restriction-selection and restriction-purification, deletion
mutagenesis, mutagenesis by total gene synthesis, degenerate PCR,
double-strand break repair, and many others known to persons of
skill.
[0072] Optionally, mutagenesis can be guided by known information
from a naturally occurring polymerase molecule, or of a known
altered or mutated polymerase (e.g., using an existing mutant
polymerase that displays reduced exonuclease activity), e.g.,
sequence, sequence comparisons, physical properties, crystal
structure and/or the like as discussed above. However, in another
class of embodiments, modification can be essentially random (e.g.,
as in classical DNA shuffling).
[0073] Additional information on mutation formats is found in:
Sambrook et al., Molecular Cloning--A Laboratory Manual (3rd Ed.),
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
2000 ("Sambrook"); Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(supplemented through 2006) ("Ausubel")) and PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis). The following publications and
references cited within provide still additional detail on mutation
formats: Arnold, Protein engineering for unusual environments,
Current Opinion in Biotechnology 4:450-455 (1993); Bass et al.,
Mutant Trp repressors with new DNA-binding specificities, Science
242:240-245 (1988); Botstein & Shortle, Strategies and
applications of in vitro mutagenesis, Science 229:1193-1201 (1985);
Carter et al., Improved oligonucleotide site-directed mutagenesis
using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter,
Site-directed mutagenesis, Biochem. J. 237:1-7 (1986); Carter,
Improved oligonucleotide-directed mutagenesis using M13 vectors,
Methods in Enzymol. 154: 382-403 (1987); Dale et al.,
Oligonucleotide-directed random mutagenesis using the
phosphorothioate method, Methods Mol. Biol. 57:369-374 (1996);
Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate
large deletions, Nucl. Acids Res. 14: 5115 (1986); Fritz et al.,
Oligonucleotide-directed construction of mutations: a gapped duplex
DNA procedure without enzymatic reactions in vitro, Nucl. Acids
Res. 16: 6987-6999 (1988); Grundstrom et al.,
Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Kunkel, The
efficiency of oligonucleotide directed mutagenesis, in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapid and efficient
site-specific mutagenesis without phenotypic selection, Proc. Natl.
Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid and
efficient site-specific mutagenesis without phenotypic selection,
Methods in Enzymol. 154, 367-382 (1987); Kramer et al., The gapped
duplex DNA approach to oligonucleotide-directed mutation
construction, Nucl. Acids Res. 12: 9441-9456 (1984); Kramer &
Fritz Oligonucleotide-directed construction of mutations via gapped
duplex DNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al.,
Point Mismatch Repair, Cell 38:879-887 (1984); Kramer et al.,
Improved enzymatic in vitro reactions in the gapped duplex DNA
approach to oligonucleotide-directed construction of mutations,
Nucl. Acids Res. 16: 7207 (1988); Ling et al., Approaches to DNA
mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997);
Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki,
Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis, Proc.
Natl. Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein,
Inhibition of restriction endonuclease Nci I cleavage by
phosphorothioate groups and its application to
oligonucleotide-directed mutagenesis, Nucl. Acids Res. 14:
9679-9698 (1986); Nambiar et al., Total synthesis and cloning of a
gene coding for the ribonuclease S protein, Science 223: 1299-1301
(1984); Sakamar and Khorana, Total synthesis and expression of a
gene for the a-subunit of bovine rod outer segment guanine
nucleotide-binding protein (transducin), Nucl. Acids Res. 14:
6361-6372 (1988); Sayers et al., Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl.
Acids Res. 16:791-802 (1988); Sayers et al., Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide,
(1988) Nucl. Acids Res. 16: 803-814; Sieber, et al., Nature
Biotechnology, 19:456-460 (2001); Smith, In vitro mutagenesis, Ann.
Rev. Genet. 19:423-462 (1985); Methods in Enzymol. 100: 468-500
(1983); Methods in Enzymol. 154: 329-350 (1987); Stemmer, Nature
370, 389-91 (1994); Taylor et al., The use of
phosphorothioate-modified DNA in restriction enzyme reactions to
prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor
et al., The rapid generation of oligonucleotide-directed mutations
at high frequency using phosphorothioate-modified DNA, Nucl. Acids
Res. 13: 8765-8787 (1985); Wells et al., Importance of
hydrogen-bond formation in stabilizing the transition state of
subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells
et al., Cassette mutagenesis: an efficient method for generation of
multiple mutations at defined sites, Gene 34:315-323 (1985); Zoller
& Smith, Oligonucleotide-directed mutagenesis using M13-derived
vectors: an efficient and general procedure for the production of
point mutations in any DNA fragment, Nucleic Acids Res.
10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed
mutagenesis of DNA fragments cloned into M13 vectors, Methods in
Enzymol. 100:468-500 (1983); and Zoller & Smith,
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template, Methods
in Enzymol. 154:329-350 (1987). Additional details on many of the
above methods can be found in Methods in Enzymology Volume 154,
which also describes useful controls for trouble-shooting problems
with various mutagenesis methods.
[0074] Determining Kinetic Parameters
[0075] The polymerases of the invention can be screened or
otherwise tested to determine whether the polymerase displays a
modified activity for or with a nucleotide analogue as compared to
the first DNA polymerase (e.g., a corresponding wild-type
polymerase from which the recombinant polymerase was derived). For
example, k.sub.cat, K.sub.m, V.sub.max, k.sub.cat/K.sub.m,
V.sub.max/K.sub.m, k.sub.pol, and/or K.sub.d of the recombinant DNA
polymerase for the nucleotide analogue can be determined. Further,
k.sub.cat, K.sub.m, V.sub.max, V.sub.max/K.sub.m,
k.sub.cat/K.sub.m, k.sub.pol, and/or K.sub.d of the recombinant DNA
polymerase for a natural nucleotide can also be determined (e.g.,
where the polymerase desirably includes both analogue and natural
nucleotide incorporation activity).
[0076] As is well-known in the art, for enzymes obeying simple
Michaelis-Menten kinetics, kinetic parameters are readily derived
from rates of catalysis measured at different substrate
concentrations. The Michaelis-Menten equation,
V=V.sub.max[S]([S]+K.sub.m).sup.-1, relates the concentration of
uncombined substrate ([S], approximated by the total substrate
concentration), the maximal rate (V.sub.max, attained when the
enzyme is saturated with substrate), and the Michaelis constant
(K.sub.m, equal to the substrate concentration at which the
reaction rate is half of its maximal value), to the reaction rate
(V).
[0077] For many enzymes, K.sub.m is equal to the dissociation
constant of the enzyme-substrate complex and is thus a measure of
the strength of the enzyme-substrate complex. For such an enzyme,
in a comparison of K.sub.ms, a lower K.sub.m represents a complex
with stronger binding, while a higher Km represents a complex with
weaker binding. The ratio k.sub.cat/K.sub.m, sometimes called the
specificity constant, represents the apparent rate constant for
combination of substrate with free enzyme. The larger the
specificity constant, the more efficient the enzyme is in binding
the substrate and converting it to product.
[0078] The k.sub.cat (also called the turnover number of the
enzyme) can be determined if the total enzyme concentration
([E.sub.T], i.e., the concentration of active sites) is known,
since V.sub.max=k.sub.cat[E.sub.T]. For situations in which the
total enzyme concentration is difficult to measure, the ratio
V.sub.max/K.sub.m is often used instead as a measure of efficiency.
K.sub.m and V.sub.max can be determined, for example, from a
Lineweaver-Burk plot of 1/V against 1/[S], where the y intercept
represents 1/V.sub.max, the x intercept -1/K.sub.m, and the slope
K.sub.m/V.sub.max, or from an Eadie-Hofstee plot of V against
V/[S], where the y intercept represents V.sub.max, the x intercept
V.sub.max/K.sub.m, and the slope -K.sub.m. Software packages such
as KinetAsyst.TM. or Enzfit (Biosoft, Cambridge, UK) can facilitate
the determination of kinetic parameters from catalytic rate
data.
[0079] For enzymes such as polymerases that have multiple
substrates, varying the concentration of only one substrate while
holding the others in suitable excess (e.g., effectively constant)
concentration typically yields normal Michaelis-Menten
kinetics.
[0080] In one embodiment, using presteady-state kinetics, the
nucleotide concentration dependence of the rate k.sub.obs (the
observed first-order rate constant for dNTP incorporation) provides
an estimate of the K.sub.m for a ground state binding and the
maximum rate of polymerization (k.sub.w). The k.sub.obs is measured
using a burst assay. The results of the assay are fitted with the
burst equation; Product=A[1-exp(-k.sub.obs*t)]+k.sub.ss*t where A
represents amplitude an estimate of the concentration of the enzyme
active sites, k.sub.ss is the observed steady-state rate constant
and t is the reaction incubation time. The K.sub.m for dNTP binding
to the polymerase-DNA complex and the k.sub.pol are calculated by
fitting the dNTP concentration dependent change in the k.sub.obs
using the equation k.sub.obs=(k.sub.pol*[S])*(K.sub.m+[S])-1 where
[S] is the substrate concentration. Results are optionally obtained
from a rapid-quench experiment (also called a quench-flow
measurement), for example, based on the methods described in
Johnson (1986) "Rapid kinetic analysis of mechanochemical
adenosinetriphosphatases" Methods Enzymol. 134:677-705, Patel et
al. (1991) "Pre-steady-state kinetic analysis of processive DNA
replication including complete characterization of an
exonuclease-deficient mutant" Biochemistry 30(2):511-25, and Tsai
and Johnson (2006) "A new paradigm for DNA polymerase specificity"
Biochemistry 45(32):9675-87.
[0081] Parameters such as rate of binding of a nucleotide analogue
by the recombinant polymerase, rate of product release by the
recombinant polymerase, or branching rate of the recombinant
polymerase (the "branching rate" is the rate of dissociation of a
nucleotide or nucleotide analogue from the polymerase active site
without incorporation of the nucleotide or nucleotide analogue,
where the nucleotide or nucleotide analogue if it were incorporated
would correctly base-pair with a complementary nucleotide or
nucleotide analogue in the template) can also be determined, and
optionally compared to that of the first polymerase (e.g., a
corresponding wild-type polymerase). See, e.g., Example 3
herein.
[0082] For a more thorough discussion of enzyme kinetics, see,
e.g., Berg, Tymoczko, and Stryer (2002) Biochemistry, Fifth
Edition, W. H. Freeman; Creighton (1984) Proteins: Structures and
Molecular Principles, W. H. Freeman; and Fersht (1985) Enzyme
Structure and Mechanism, Second Edition, W. H. Freeman.
[0083] As discussed above, the relevant DNA polymerase has a
modified active site region that is homologous to a wild-type
active site region of a wild-type DNA polymerase e.g., that
includes one or more structural modification relative to the wild
type active site region that increases the relative activity of the
enzyme to one or more of natural nucleotides and/or nucleotide
analogues, with increases in activity to nucleotide analogues being
a preferred goal. In at least one aspect, without being bound to
any particular theory of operation, the modifications are targeted
to reduce steric inhibition for entry of the nucleotide analogue
into the modified active site and/or that is complementary with one
or more non-natural features of the nucleotide analogue. A K.sub.m
value of the recombinant polymerase for the nucleotide analogue is
typically lower than a K.sub.m for a corresponding homologous
wild-type polymerase for the nucleotide analogue.
[0084] In one aspect, the improved activity of the enzymes of the
invention is measured with reference to a model analogue or
analogue set and compared with a given parental enzyme. For
example, in the case of enzymes derived from a .PHI.29 parental
enzyme, an improved enzyme of the invention would have a lower Km
than the parental enzyme, e.g., wild type D29 or N62D .PHI.29,
toward a given analogue. In general, for purposes of discussion,
examples of improved enzymes of the invention will be
characterizable as having lower K.sub.ms toward A488dC4P and/or
A568dC4P, two analogs that have been reasonably well processed and
reasonably poorly processed by .PHI.29 derived enzymes,
respectively, that are, e.g., from about 5% or less to about 90% or
less of the Km possessed by N62D .PHI.29 toward the same analogs.
For example, as set forth in more detail in the examples below,
e.g., at Table 2, His-375H-N62D .PHI.29 (SEQ ID NO:4) displays a
K.sub.m that is about 40% of K.sub.m of N62D .PHI.29 (SEQ ID NO:2)
for A488dC4P, while His-375S-N62D .PHI.29 (SEQ ID NO:5) displays a
K.sub.m that is about 75% of the K.sub.m of N62D .PHI.29 (SEQ ID
NO:2) for A488dC4P. Similarly, His-375H-N62D .PHI.29 (SEQ ID NO:4)
displays a K.sub.m that is about 15% of the K.sub.m of N62D .PHI.29
(SEQ ID NO:2) for A568dC4P, while His-375S-N62D .PHI.29 (SEQ ID
NO:5) displays a K.sub.m that is about 38% of the K.sub.m of N62D
.PHI.29 (SEQ ID NO:2) for A568dC4P. While the foregoing may be used
as a characterization tool, it in no way is intended as a
specifically limiting reaction of the invention.
[0085] Screening Polymerases
[0086] Screening or other protocols can be used to determine
whether a polymerase displays a modified activity for a nucleotide
analogue as compared to the first DNA polymerase. For example,
k.sub.cat, K.sub.m, V.sub.max, or k.sub.cat/K.sub.m of the
recombinant DNA polymerase for the nucleotide analogue can be
determined as discussed above. Further, k.sub.cat, K.sub.m,
V.sub.max, or k.sub.cat/K.sub.m of the recombinant DNA polymerase
for a natural nucleotide can also be similarly determined (e.g.,
where the polymerase desirably includes both analogue and natural
nucleotide incorporation activity).
[0087] In one desirable aspect, a library of recombinant DNA
polymerases can be made and screened for these properties. For
example, a plurality of members of the library can be made to
include one or more putative steric inhibition feature mutation
an/or a mutation to putatively produce complementary with one or
more non-natural features of the nucleotide analogue, that is then
screened for the properties of interest. In general, the library
can be screened to identify at least one member comprising a
modified activity of interest.
[0088] Libraries of polymerases can be either physical or logical
in nature. Moreover, any of a wide variety of library formats can
be used. For example, polymerases can be fixed to solid surfaces in
arrays of proteins. Similarly, liquid phase arrays of polymerases
(e.g., in microwell plates) can be constructed for convenient
high-throughput fluid manipulations of solutions comprising
polymerases. Liquid, emulsion, or gel-phase libraries of cells that
express recombinant polymerases can also be constructed, e.g., in
microwell plates, or on agar plates. Phage display libraries of
polymerases or polymerase domains (e.g., including the active site
region) can be produced. Instructions in making and using libraries
can be found, e.g., in Sambrook, Ausubel and Berger, referenced
herein.
[0089] For the generation of libraries involving fluid transfer to
or from microtiter plates, a fluid handling station is optionally
used. Several "off the shelf" fluid handling stations for
performing such transfers are commercially available, including
e.g., the Zymate systems from Caliper Life Sciences (Hopkinton,
Mass.) and other stations which utilize automatic pipettors, e.g.,
in conjunction with the robotics for plate movement (e.g., the
ORCA.RTM. robot, which is used in a variety of laboratory systems
available, e.g., from Beckman Coulter, Inc. (Fullerton,
Calif.).
[0090] In an alternate embodiment, fluid handling is performed in
microchips, e.g., involving transfer of materials from microwell
plates or other wells through microchannels on the chips to
destination sites (microchannel regions, wells, chambers or the
like). Commercially available microfluidic systems include those
from Hewlett-Packard/Agilent Technologies (e.g., the HP2100
bioanalyzer) and the Caliper High Throughput Screening System. The
Caliper High Throughput Screening System provides one example
interface between standard microwell library formats and Labchip
technologies. Furthermore, the patent and technical literature
includes many examples of microfluidic systems which can interface
directly with microwell plates for fluid handling.
[0091] Desirable Properties
[0092] The polymerases of the invention can include any of a
variety of modified properties towards natural or nucleotide
analogues or analogues, depending on the application, including
increased speed, increased retention time (or decreased speed) for
incorporated bases, greater processivity, etc. For example, where a
higher level of nucleotide analogue incorporation is desired, the
polymerase of the invention is selected to have a lower K.sub.m, a
higher Vmax and/or a higher k.sub.cat than a corresponding
homologous wild-type polymerase with respect to a given nucleotide
analogue. In certain embodiments, it is desirable to slow or
quicken the overall nucleotide incorporation speed of the
polymerase (e.g., depending on the resolution of the equipment used
to monitor incorporation), or to improve processivity, specificity,
or the like. In certain embodiments, the recombinant polymerase has
an increased rate of binding of a nucleotide analogue, an increased
rate of product release, and/or a decreased branching rate, as
compared to a corresponding homologous wild-type polymerase. Any of
these features can be screened for or against in selecting a
polymerase of the invention.
[0093] For example, the polymerases of the invention can typically
incorporate natural nucleotides (e.g., A, C, G and T) into a
growing copy nucleic acid. For example, the recombinant polymerase
optionally displays a specific activity for a natural nucleotide
that is at least about 5% as high (e.g., 5%, 10%, 25%, 50%, 75%,
100% or higher) as a corresponding homologous wild-type polymerase
and a processivity with natural nucleotides in the presence of a
template that is at least 5% as high (e.g., 5%, 10%, 25%, 50%, 75%,
100% or higher) as the wild-type polymerase in the presence of the
natural nucleotide. Optionally, the recombinant polymerase also
displays a k.sub.cat/K.sub.m or V.sub.max/K.sub.m for a naturally
occurring nucleotide that is at least about 10% as high (e.g., 10%,
25%, 50%, 75% or 100% or higher) as the wild-type polymerase.
[0094] Additional Example Details
[0095] A number of specific examples of modified active site
regions are described herein. An "active site region" is a portion
of the polymerase that includes or is proximal to the active site
(e.g., within about 2 nm of the active site) in a three dimensional
structure of a folded polymerase. Specific examples of structural
modifications within or proximal to the active site of .PHI.29 DNA
polymerase are described herein. For example, relative to a
wild-type .PHI.29 DNA polymerase, these modification can include
any of: a deletion of .DELTA.505-525, a deletion within the
.DELTA.505-525 domain, a K135A mutation, an L384R mutation (e.g.,
in combination with another mutation herein), an E375H mutation, an
E375S mutation, an E375K mutation, an E375R mutation, an E375A
mutation, an E375Q mutation, an E375W mutation, an E375Y mutation,
an E375F mutation, an E486A mutation, an E486D mutation, a K512A
mutation, a mutation listed in Table 8, and combinations thereof.
For example, the polymerase can include a combination of mutations
selected from the list of combinations in Table 8.
[0096] The polymerase optionally further includes one or more
mutations/deletions relative to the wild-type polymerase that
reduce or eliminate endogenous exonuclease activity. For example,
relative to the wild-type .PHI.29 DNA polymerase, N62 is optionally
mutated or deleted to reduce exonuclease activity; e.g., the
polymerase can include an N62D mutation. Other example mutations
that reduce exonuclease activity include D12A, T15I, E141, and/or
D66A; accordingly, the polymerases of the invention optionally
comprise one or more of these mutations.
[0097] As will be appreciated, the numbering of amino acid residues
is with respect to the wild-type sequence of the .PHI.29
polymerase, and actual position within a molecule of the invention
may vary based upon the nature of the various modifications that
the enzyme includes relative to the wild type .PHI.29 enzyme, e.g.,
deletions and/or additions to the molecule, either at the termini
or within the molecule itself.
[0098] Affinity Tags and Other Optional Polymerase Features
[0099] The recombinant DNA polymerase optionally includes
additional features exogenous or heterologous to the polymerase.
For example, the recombinant polymerase optionally includes one or
more exogenous affinity tags, e.g., purification or substrate
binding tags, such as a 6 His tag sequence, a GST tag, an HA tag
sequence, a plurality of 6 His tag sequences, a plurality of GST
tags, a plurality of HA tag sequences, a SNAP-tag, or the like.
These and other features useful in the context of binding a
polymerase to a surface are optionally included, e.g., to orient
and/or protect the polymerase active site when the polymerase is
bound to a surface. Other useful features include recombinant dimer
domains of the enzyme, and, e.g., large extraneous polypeptide
domains coupled to the polymerase distal to the active site. For
example, for .PHI.29, the active site is in the C terminal region
of the protein, and added surface binding elements (extra domains,
His tags, etc.) are typically located in the N-terminal region to
avoid interfering with the active site when the polymerase is
coupled to a surface.
[0100] In general, surface binding elements and purification tags
that can be added to the polymerase (recombinantly or, e.g.,
chemically) include, e.g., polyhistidine tags, HIS-6 tags, biotin,
avidin, GST sequences, BiTag sequences, S tags, SNAP-tags,
enterokinase sites, thrombin sites, antibodies or antibody domains,
antibody fragments, antigens, receptors, receptor domains, receptor
fragments, ligands, dyes, acceptors, quenchers, or combinations
thereof.
[0101] Multiple surface binding domains can be added to orient the
polypeptide relative to a surface and/or to increase binding of the
polymerase to the surface. By binding a surface at two or more
sites, through two or more separate tags, the polymerase is held in
a relatively fixed orientation with respect to the surface.
Additional details on fixing a polymerase to a surface are found in
U.S. patent application 60/753,446 "PROTEIN ENGINEERING STRATEGIES
TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS" by Hanzel et al.
and U.S. patent application 60/753,515''ACTIVE SURFACE COUPLED
POLYMERASES'' by Hanzel et al., both filed Dec. 22, 2005 and
incorporated herein by reference for all purposes, and in Attorney
Docket number 105-001210US "PROTEIN ENGINEERING STRATEGIES TO
OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS" by Hanzel et al.
and Attorney docket 105-00810US "ACTIVE SURFACE COUPLED
POLYMERASES" by Hanzel et al. both co-filed herewith and
incorporated herein by reference for all purposes.
Applications for Enhanced Incorporation of Nucleotide Analogues by
a DNA Polymerase
[0102] Polymerases of the invention, natural and/or nucleotide
analogues and nucleic acid templates (DNA or RNA) are optionally
used to copy the template nucleic acid. That is, a mixture of the
polymerase, nucleotide analogues, and optionally natural
nucleotides and other reagents, the template and a replication
initiating moiety is reacted such that the polymerase extends the
primer in a template-dependent manner. The moiety can be a standard
oligonucleotide primer, or, alternatively, a component of the
template, e.g., the template can be a self-priming single stranded
DNA, a nicked double stranded DNA, or the like. Similarly, a
terminal protein can serve as a initiating moiety. At least one
nucleotide analogue can be incorporated into the DNA. The template
DNA can be a linear or circular DNA, and in certain applications,
is desirably a circular template (e.g., for rolling circle
replication or for sequencing of circular templates). Optionally,
the composition can be present in an automated DNA replication
and/or sequencing system.
[0103] Incorporation of labeled nucleotide analogues by the
polymerases of the invention are particularly useful in a variety
of different nucleic acid analyses, including real-time monitoring
of DNA polymerization. The label can itself be incorporated, or
more preferably, can be released during incorporation. For example,
analogue incorporation can be monitored in real-time by monitoring
label release during incorporation of the analogue by the
polymerase. The portion of the analogue that is incorporated can be
the same as a natural nucleotide, or can include features of the
analogue that differ from a natural nucleotide.
[0104] In general, label incorporation or release can be used to
indicate the presence and composition of a growing nucleic acid
strand, e.g., providing evidence of template
replication/amplification and/or sequence of the template.
Signaling from the incorporation can be the result of detecting
labeling groups that are liberated from the incorporated analogue,
e.g., in a solid phase assay, or can arise upon the incorporation
reaction. For example, in the case of FRET labels where a bound
label is quenched and a free label is not, release of a label group
from the incorporated analogue can give rise to a fluorescent
signal. Alternatively, the enzyme may be labeled with one member of
a FRET pair proximal to the active site, and incorporation of an
analogue bearing the other member will allow energy transfer upon
incorporation. The use of enzyme bound FRET components in nucleic
acid sequencing applications is described, e.g., in Published U.S.
Patent application No. 2003-0044781, incorporated herein by
reference.
[0105] In one example reaction of interest, a polymerase reaction
can be isolated within an extremely small observation volume that
effectively results in observation of individual polymerase
molecules. As a result, the incorporation event provides
observation of an incorporating nucleotide analogue that is readily
distinguishable from non-incorporated nucleotide analogs. In a
preferred aspect, such small observation volumes are provided by
immobilizing the polymerase enzyme within an optical confinement,
such as a Zero Mode Waveguide. For a description of ZMWs and their
application in single molecule analyses, and particularly nucleic
acid sequencing, see, e.g., Published U.S. Patent Application No.
2003/0044781, and U.S. Pat. No. 6,917,726, each of which is
incorporated herein by reference in its entirety for all
purposes.
[0106] In general, a polymerase enzyme is complexed with the
template strand in the presence of one or more nucleotides and/or
one or more nucleotide analogue of the invention. For example, in
certain embodiments, labeled analogues are present representing
analogous compounds to each of the four natural nucleotides, A, T,
G and C, e.g., in separate polymerase reactions, as in classical
Sanger sequencing, or multiplexed together in a single reaction, as
in multiplexed sequencing approaches. When a particular base in the
template strand is encountered by the polymerase during the
polymerization reaction, it complexes with an available analogue
that is complementary to such nucleotide, and incorporates that
analogue into the nascent and growing nucleic acid strand. In one
aspect, incorporation can result in a label being released, e.g.,
in polyphosphate analogues, cleaving between the .alpha. and .beta.
phosphorus atoms in the analogue, and consequently releasing the
labeling group (or a portion thereof). The incorporation event is
detected, either by virtue of a longer presence of the analogue
and, thus, the label, in the complex, or by virtue of release of
the label group into the surrounding medium. Where different
labeling groups are used for each of the types of analogs, e.g., A,
T, G or C, identification of a label of an incorporated analogue
allows identification of that analogue and consequently,
determination of the complementary nucleotide in the template
strand being processed at that time. Sequential reaction and
monitoring permits a real-time monitoring of the polymerization
reaction and determination of the sequence of the template nucleic
acid. As noted above, in particularly preferred aspects, the
polymerase enzyme/template complex is provided immobilized within
an optical confinement that permits observation of an individual
complex, e.g., a Zero Mode Waveguide. In addition to their use in
sequencing, the analogs of the invention are also equally useful in
a variety of other genotyping analyses, e.g., SNP genotyping using
single base extension methods, real time monitoring of
amplification, e.g., RT-PCR methods, and the like.
[0107] Further details regarding sequencing and nucleic acid
amplification can be found in Sambrook et al., Molecular Cloning--A
Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook"); Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2006)
("Ausubel")) and PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
("Innis").
Making and Isolating Recombinant Polymerases
[0108] Generally, nucleic acids encoding a polymerase of the
invention can be made by cloning, recombination, in vitro
synthesis, in vitro amplification and/or other available methods. A
variety of recombinant methods can be used for expressing an
expression vector that encodes a polymerase of the invention, e.g.,
a mutant polymerase that, without being bound to a particular
theory, reduces steric hindrance for a nucleotide analogue of the
invention and/or that includes a complementarity feature.
Recombinant methods for making nucleic acids, expression and
isolation of expressed products are described, e.g., in Sambrook,
Ausubel and Innis.
[0109] In addition, a plethora of kits are commercially available
for the purification of plasmids or other relevant nucleic acids
from cells, (see, e.g., EasyPrep.TM., FlexiPrep.TM., both from
Pharmacia Biotech; StrataClean.TM., from Stratagene; and,
QIAprep.TM. from Qiagen). Any isolated and/or purified nucleic acid
can be further manipulated to produce other nucleic acids, used to
transfect cells, incorporated into related vectors to infect
organisms for expression, and/or the like. Typical cloning vectors
contain transcription and translation terminators, transcription
and translation initiation sequences, and promoters useful for
regulation of the expression of the particular target nucleic acid.
The vectors optionally comprise generic expression cassettes
containing at least one independent terminator sequence, sequences
permitting replication of the cassette in eukaryotes, or
prokaryotes, or both, (e.g., shuttle vectors) and selection markers
for both prokaryotic and eukaryotic systems. Vectors are suitable
for replication and integration in prokaryotes, eukaryotes, or
both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al.,
Nature, 328:731 (1987); Schneider, B., et al., Protein Expr. Purif.
6435:10 (1995); Ausubel, Sambrook, Berger (above). A catalogue of
Bacteria and Bacteriophages useful for cloning is provided, e.g.,
by the ATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage
published yearly by the ATCC. Additional basic procedures for
sequencing, cloning and other aspects of molecular biology and
underlying theoretical considerations are also found in Watson et
al. (1992) Recombinant DNA Second Edition, Scientific American
Books, NY.
[0110] In addition, systems of orthogonal components are available
that can incorporate any of a variety of unnatural amino acids into
a recombinant protein (e.g., polymerase of the invention). In
brief, a cell or other translation system (e.g., an in vitro
translation system) is constructed that includes an orthogonal tRNA
("OtRNA"; a tRNA not recognized by the cell's endogenous
translation machinery, such as an amber or 4-base tRNA) and an
orthogonal tRNA synthetase ("ORS"; this is a synthetase that does
not aminoacylate any endogenous tRNA of the cell, but which can
aminoacylate the OtRNA in response to a selector codon). A nucleic
acid encoding the enzyme is constructed to include a selector codon
at a selected site that is specifically recognized by the OtRNA.
The ORS specifically incorporates an unnatural amino acid with a
desired chemical functionality at one or more selected site(s)
(e.g., distal to the active site). This chemical functional group
can be unique as compared to those ordinarily found on amino acids,
e.g., that incorporate keto or other functionalities. Further
information on orthogonal systems can be found, e.g., in Wang et
al., (2001), Science 292:498-500; Chin et al., (2002) Journal of
the American Chemical Society 124:9026-9027; Chin and Schultz,
(2002), ChemBioChem 11:1135-1137; Chin, et al., (2002), PNAS United
States of America 99:11020-11024; and Wang and Schultz, (2002),
Chem. Comm., 1-10. See also, International Publications WO
2002/086075, entitled "METHODS AND COMPOSITIONS FOR THE PRODUCTION
OF ORTHOGONAL tRNA AMINOACYL-tRNA SYNTHETASE PAIRS;" WO
2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINO
ACIDS;" WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC
CODE;" WO 2005/019415, filed Jul. 7, 2004; WO 2005/007870, filed
Jul. 7, 2004; and WO 2005/007624, filed Jul. 7, 2004.
[0111] Other useful references, e.g. for cell isolation and culture
(e.g., for subsequent nucleic acid isolation) include Freshney
(1994) Culture of Animal Cells, a Manual of Basic Technique, third
edition, Wiley-Liss, New York and the references cited therein;
Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems
John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips
(eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental
Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New
York) and Atlas and Parks (eds) The Handbook of Microbiological
Media (1993) CRC Press, Boca Raton, Fla.
[0112] In addition, essentially any nucleic acid can be custom or
standard ordered from any of a variety of commercial sources, such
as Operon Technologies Inc. (Alameda, Calif.).
[0113] A variety of protein isolation and detection methods are
known and can be used to isolate polymerases, e.g., from
recombinant cultures of cells expressing the recombinant
polymerases of the invention. A variety of protein isolation and
detection methods are well known in the art, including, e.g., those
set forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y.
(1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification, Academic Press, Inc. N.Y. (1990); Sandana (1997)
Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.
(1996) Protein Methods, 2.sup.nd Edition Wiley-Liss, NY; Walker
(1996) The Protein Protocols Handbook Humana Press, NJ, Harris and
Angal (1990) Protein Purification Applications: A Practical
Approach IRL Press at Oxford, Oxford, England; Harris and Angal
Protein Purification Methods: A Practical Approach IRL Press at
Oxford, Oxford, England; Scopes (1993) Protein Purification:
Principles and Practice 3.sup.rd Edition Springer Verlag, NY;
Janson and Ryden (1998) Protein Purification: Principles, High
Resolution Methods and Applications, Second Edition Wiley-VCH, NY;
and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and
the references cited therein. Additional details regarding protein
purification and detection methods can be found in Satinder Ahuja
ed., Handbook of Bioseparations, Academic Press (2000).
Kits
[0114] The present invention also provides kits that incorporate
the polymerases of the invention, e.g., with one or more nucleotide
analogues, e.g., for sequencing, nucleic acid amplification, or the
like. Such kits can include the polymerase of the invention
packaged in a fashion to enable use of the polymerase, a set of
different nucleotide analogs of the invention, e.g., those that are
analogous to A, T, G, and C, e.g., where at least one of the
analogues bears a detectable moiety, and in preferred aspects more
than one, and in many cases, each bears a detectably different
labeling group, optionally to permit identification in the presence
of the other analogues. Depending upon the desired application, the
kits of the invention optionally include additional reagents, such
as natural nucleotides, a control template, and other reagents,
such as buffer solutions and/or salt solutions, including, e.g.,
divalent metal ions, i.e., Mg.sup.++, Mn.sup.++ and/or Fe.sup.++,
standard solutions, e.g., dye standards for detector calibration.
Such kits also typically include instructions for use of the
compounds and other reagents in accordance with the desired
application methods, e.g., nucleic acid sequencing, amplification
and the like.
Nucleic Acid and Polypeptide Sequence and Variants
[0115] As described herein, the invention provides polynucleotide
sequences encoding, e.g., a polymerase as described herein.
Examples of polymerase sequences that include steric hindrance or
complementarity features are found herein, e.g., in Table 3.
However, one of skill in the art will immediately appreciate that
the invention is not limited to those sequences. For example, one
of skill will appreciate that the invention also provides, e.g.,
many related sequences with the functions described herein, e.g.,
polynucleotides and polypeptides encoding conservative variants of
a polymerase of Table 3.
[0116] Accordingly, the invention provides a variety of
polypeptides (polymerases) and polynucleotides (nucleic acids that
encode polymerases). Example polynucleotides of the invention
include, e.g., a polynucleotide comprising a nucleotide sequence as
set forth in Table 3 or a polynucleotide that is complementary to
or that encodes a polynucleotide sequence thereof (e.g., where the
given sequence is a DNA, an RNA is one example of a sequence that
encodes the DNA, e.g., via reverse transcription). A polynucleotide
of the invention also optionally includes any polynucleotide that
encodes a polymerase of Table 3. Because of the degeneracy of the
genetic code, many polynucleotides equivalently encode a given
polymerase sequence. Similarly, an artificial or recombinant
nucleic acid that hybridizes to a polynucleotide indicated above
under highly stringent conditions over substantially the entire
length of the nucleic acid (and is other than a naturally occurring
polynucleotide) is a polynucleotide of the invention. In one
embodiment, a composition includes a polypeptide of the invention
and an excipient (e.g., buffer, water, pharmaceutically acceptable
excipient, etc.). The invention also provides an antibody or
antisera specifically immunoreactive with a polypeptide of the
invention (e.g., that specifically recognizes an altered steric
hindrance or nucleotide analogue complementarity feature.
[0117] In certain embodiments, a vector (e.g., a plasmid, a cosmid,
a phage, a virus, etc.) comprises a polynucleotide of the
invention. In one embodiment, the vector is an expression vector.
In another embodiment, the expression vector includes a promoter
operably linked to one or more of the polynucleotides of the
invention. In another embodiment, a cell comprises a vector that
includes a polynucleotide of the invention.
[0118] One of skill will also appreciate that many variants of the
disclosed sequences are included in the invention. For example,
conservative variations of the disclosed sequences that yield a
functionally similar sequence are included in the invention.
Variants of the nucleic acid polynucleotide sequences, wherein the
variants hybridize to at least one disclosed sequence, are
considered to be included in the invention. Unique subsequences of
the sequences disclosed herein, as determined by, e.g., standard
sequence comparison techniques, are also included in the
invention.
[0119] Conservative Variations
[0120] Owing to the degeneracy of the genetic code, "silent
substitutions" (i.e., substitutions in a nucleic acid sequence
which do not result in an alteration in an encoded polypeptide) are
an implied feature of every nucleic acid sequence that encodes an
amino acid sequence. Similarly, "conservative amino acid
substitutions," where one or a limited number of amino acids in an
amino acid sequence are substituted with different amino acids with
highly similar properties, are also readily identified as being
highly similar to a disclosed construct. Such conservative
variations of each disclosed sequence are a feature of the present
invention.
[0121] "Conservative variations" of a particular nucleic acid
sequence refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or, where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. One of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 4%, 2% or 1%) in an encoded
sequence are "conservatively modified variations" where the
alterations result in the deletion of an amino acid, addition of an
amino acid, or substitution of an amino acid with a chemically
similar amino acid, while retaining the relevant reduced steric
hindrance or nucleotide analogue complementarity feature (for
example, the conservative substitution can be of a residue distal
to the active site region). Thus, "conservative variations" of a
listed polypeptide sequence of the present invention include
substitutions of a small percentage, typically less than 5%, more
typically less than 2% or 1%, of the amino acids of the polypeptide
sequence, with an amino acid of the same conservative substitution
group. Finally, the addition of sequences which do not alter the
encoded activity of a nucleic acid molecule, such as the addition
of a non-functional or tagging sequence (introns in the nucleic
acid, poly His or similar sequences in the encoded polypeptide,
etc.), is a conservative variation of the basic nucleic acid or
polypeptide.
[0122] In one aspect, the conservative substitution includes one or
more deletion or substitution of a residue at an amino acid residue
of the polymerase corresponding to amino acid residue 375.
[0123] Conservative substitution tables providing functionally
similar amino acids are well known in the art, where one amino acid
residue is substituted for another amino acid residue having
similar chemical properties (e.g., aromatic side chains or
positively charged side chains), and therefore does not
substantially change the functional properties of the polypeptide
molecule. The following sets forth example groups that contain
natural amino acids of like chemical properties, where
substitutions within a group is a "conservative substitution".
TABLE-US-00001 TABLE A Conservative Amino Acid Substitutions
Nonpolar and/or Negatively Aliphatic Polar, Positively Charged Side
Uncharged Aromatic Side Charged Side Side Chains Side Chains Chains
Chains Chains Glycine Serine Phenylalanine Lysine Aspartate Alanine
Threonine Tyrosine Arginine Glutamate Valine Cysteine Tryptophan
Histidine Leucine Methionine Isoleucine Asparagine Proline
Glutamine
[0124] Nucleic Acid Hybridization
[0125] Comparative hybridization can be used to identify nucleic
acids of the invention, including conservative variations of
nucleic acids of the invention. In addition, target nucleic acids
which hybridize to a nucleic acid represented in Table 3 under
high, ultra-high and ultra-ultra high stringency conditions, where
the nucleic acids are other than a naturally occurring .PHI.29, or
an N62D mutant, are a feature of the invention. Examples of such
nucleic acids include those with one or a few silent or
conservative nucleic acid substitutions as compared to a given
nucleic acid sequence of Table 3.
[0126] A test nucleic acid is said to specifically hybridize to a
probe nucleic acid when it hybridizes at least 50% as well to the
probe as to the perfectly matched complementary target, i.e., with
a signal to noise ratio at least half as high as hybridization of
the probe to the target under conditions in which the perfectly
matched probe binds to the perfectly matched complementary target
with a signal to noise ratio that is at least about
5.times.-10.times. as high as that observed for hybridization to
any of the unmatched target nucleic acids.
[0127] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of well
characterized physico-chemical forces, such as hydrogen bonding,
solvent exclusion, base stacking and the like. An extensive guide
to the hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes part I chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, New York), as well as in
Current Protocols in Molecular Biology, Ausubel et al., eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through 2004) ("Ausubel"); Hames and Higgins (1995) Gene Probes 1
IRL Press at Oxford University Press, Oxford, England, (Hames and
Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at
Oxford University Press, Oxford, England (Hames and Higgins 2)
provide details on the synthesis, labeling, detection and
quantification of DNA and RNA, including oligonucleotides.
[0128] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formalin with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of
stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C.
for 15 minutes (see, Sambrook, supra for a description of SSC
buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example low
stringency wash is 2.times.SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 5.times. (or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0129] "Stringent hybridization wash conditions" in the context of
nucleic acid hybridization experiments such as Southern and
northern hybridizations are sequence dependent, and are different
under different environmental parameters. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993), supra.
and in Hames and Higgins, 1 and 2. Stringent hybridization and wash
conditions can easily be determined empirically for any test
nucleic acid. For example, in determining stringent hybridization
and wash conditions, the hybridization and wash conditions are
gradually increased (e.g., by increasing temperature, decreasing
salt concentration, increasing detergent concentration and/or
increasing the concentration of organic solvents such as formalin
in the hybridization or wash), until a selected set of criteria are
met. For example, in highly stringent hybridization and wash
conditions, the hybridization and wash conditions are gradually
increased until a probe binds to a perfectly matched complementary
target with a signal to noise ratio that is at least 5.times. as
high as that observed for hybridization of the probe to an
unmatched target.
[0130] "Very stringent" conditions are selected to be equal to the
thermal melting point (T.sub.m) for a particular probe. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the test sequence hybridizes to a perfectly matched probe.
For the purposes of the present invention, generally, "highly
stringent" hybridization and wash conditions are selected to be
about 5.degree. C. lower than the T.sub.m for the specific sequence
at a defined ionic strength and pH.
[0131] "Ultra high-stringency" hybridization and wash conditions
are those in which the stringency of hybridization and wash
conditions are increased until the signal to noise ratio for
binding of the probe to the perfectly matched complementary target
nucleic acid is at least 10.times. as high as that observed for
hybridization to any of the unmatched target nucleic acids. A
target nucleic acid which hybridizes to a probe under such
conditions, with a signal to noise ratio of at least 1/2 that of
the perfectly matched complementary target nucleic acid is said to
bind to the probe under ultra-high stringency conditions.
[0132] Similarly, even higher levels of stringency can be
determined by gradually increasing the hybridization and/or wash
conditions of the relevant hybridization assay. For example, those
in which the stringency of hybridization and wash conditions are
increased until the signal to noise ratio for binding of the probe
to the perfectly matched complementary target nucleic acid is at
least 10.times., 20.times., 50.times., 100.times., or 500.times. or
more as high as that observed for hybridization to any of the
unmatched target nucleic acids. A target nucleic acid which
hybridizes to a probe under such conditions, with a signal to noise
ratio of at least 1/2 that of the perfectly matched complementary
target nucleic acid is said to bind to the probe under
ultra-ultra-high stringency conditions.
[0133] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
[0134] Unique Subsequences
[0135] In some aspects, the invention provides a nucleic acid that
comprises a unique subsequence in a nucleic acid that encodes a
polymerase of Table 3. The unique subsequence may be unique as
compared to a nucleic acid corresponding to wild type (D29, or to
an N62D mutation thereof. Alignment can be performed using, e.g.,
BLAST set to default parameters. Any unique subsequence is useful,
e.g., as a probe to identify the nucleic acids of the
invention.
[0136] Similarly, the invention includes a polypeptide which
comprises a unique subsequence in a polymerase of Table 3. Here,
the unique subsequence is unique as compared to, e.g., wild type
D29, or to an N62D mutation thereof.
[0137] The invention also provides for target nucleic acids which
hybridize under stringent conditions to a unique coding
oligonucleotide which encodes a unique subsequence in a polypeptide
selected from the sequences of Table 3, wherein the unique
subsequence is unique as compared to a polypeptide corresponding to
wild type (D29, or to an N62D mutation (e.g., parental sequences
from which polymerases of the invention were derived, e.g., by
mutation). Unique sequences are determined as noted above.
[0138] Sequence Comparison, Identity, and Homology
[0139] The terms "identical" or "percent identity," in the context
of two or more nucleic acid 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, as
measured using one of the sequence comparison algorithms described
below (or other algorithms available to persons of skill) or by
visual inspection.
[0140] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides (e.g., DNAs encoding a polymerase, or
the amino acid sequence of a polymerase) refers to two or more
sequences or subsequences that have at least about 60%, about 80%,
about 90-95%, about 98%, about 99% or more nucleotide or amino acid
residue identity, when compared and aligned for maximum
correspondence, as measured using a sequence comparison algorithm
or by visual inspection. Such "substantially identical" sequences
are typically considered to be "homologous," without reference to
actual ancestry. Preferably, the "substantial identity" exists over
a region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably, the sequences are substantially
identical over at least about 150 residues, or over the full length
of the two sequences to be compared.
[0141] Proteins and/or protein sequences are "homologous" when they
are derived, naturally or artificially, from a common ancestral
protein or protein sequence. Similarly, nucleic acids and/or
nucleic acid sequences are homologous when they are derived,
naturally or artificially, from a common ancestral nucleic acid or
nucleic acid sequence. Homology is generally inferred from sequence
similarity between two or more nucleic acids or proteins (or
sequences thereof). The precise percentage of similarity between
sequences that is useful in establishing homology varies with the
nucleic acid and protein at issue, but as little as 25% sequence
similarity over 50, 100, 150 or more residues is routinely used to
establish homology. Higher levels of sequence similarity, e.g.,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be
used to establish homology. Methods for determining sequence
similarity percentages (e.g., BLASTP and BLASTN using default
parameters) are described herein and are generally available.
[0142] For sequence comparison and homology determination,
typically one sequence acts as a reference sequence to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s) relative to the reference sequence, based on the
designated program parameters.
[0143] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Current Protocols in Molecular Biology,
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
supplemented through 2004).
[0144] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. 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 then 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) and N (penalty score for
mismatching residues; always<0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. 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, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915).
[0145] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
Computer-Implemented Methods of Modeling Kinetics
[0146] In an additional aspect, the invention includes
computer-implemented methods, e.g., for modeling enzyme kinetics.
In the methods, a plurality of polymerase state transitions are
defined for discrete time steps during a template-based
polymerization reaction. In the smallest discrete time step, many
polymerase state transitions are forbidden according to the
enzymatic kinetics being modeled. A plurality of rate transition
rates are defined between the states and a multidimensional
probability matrix of possible state transitions is defined for the
smallest discrete time step, based upon a given nucleic acid
template sequence, nucleotides in a reaction mixture and the
polymerase state transitions. The resulting multidimensional
probability matrix is stored in a computer readable medium.
[0147] A variety of features of the method can vary. For example,
the polymerase state transitions are optionally user-selectable.
The transition rates between the states optionally vary depending
on nucleotide concentration, polymerase concentration, template
concentration, template sequence, position of the polymerase along
the template, characteristics of the current Watson-Crick
template-nucleotide pair, characteristics of the previous
Watson-Crick template-nucleotide pair, or characteristics of the
nucleotide being incorporated. The nucleotides in the reaction
mixture optionally comprise one or more analogue nucleotides. The
transition rates between states optionally include complete
orthogonality between every combination of multidimensional
dependencies listed above. The multidimensional probability matrix
is optionally automatically generated based upon the template
sequence, a standardized matrix of probability states, and the
nucleotides in the reaction mixture. The probability matrix is
optionally simplified by assuming that all possible Watson-Crick
base pairings are equivalent in all state transitions. The
probability matrix is further optionally simplified by assuming
that certain state transitions (eg. polymerase translocation along
DNA) are equivalent between different dimensions of the probability
matrix (eg. certain characteristics of nucleotide previously
incorporated).
[0148] Similarly, a second reagent concentration matrix is
optionally generated to account for reagent concentration changes
that result from position of the polymerase along a template, based
on an output of the probability matrix. The probability matrix is
optionally vectorized for multiple templates and the resulting
vectorized probability matrix can be multiplied by the
multidimensional probability matrix to provide a state distribution
matrix. An exponential time factor for the probability matrix can
be used to account for repeated sequences within the template
sequence. A polymerase nucleotide mismatch fraction using either a
continuum model or a counting model can be defined.
EXAMPLES
[0149] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art. Accordingly, the following examples are
offered to illustrate, but not to limit, the claimed invention.
[0150] The following sets forth a series of experiments that
demonstrate construction and characterization of a variety of
recombinant DNA polymerases having modified active site regions and
modified properties for nucleotide analogues.
Example 1
Expression of Recombinant Polymerase
[0151] A vector for expression of Phi 29 polymerase was constructed
and is schematically illustrated in FIG. 1. An N62D mutation was
introduced into wild-type Phi 29 (SEQ ID NO:1) to reduce
exonuclease activity, and GST (glutathione-S-transferase), His, and
S tags were added. The resulting tagged N62D Phi 29 amino acid
sequence is presented as SEQ ID NO:2. The sequence of the vector is
presented as SEQ ID NO:14. The tagged N62D Phi 29 polymerase is
encoded by nucleotides 4839-7428 of the vector sequence, with the
polymerase at nucleotides 5700-7428 and the N62D mutation at
nucleotides 5883-5885. Other features of the vector include the
GST-His-S tag sequences (nucleotides 4838-5699), ribosome binding
site (nucleotides 4822-4829), T7 promoter (nucleotides 4746-4758),
and kanamycin resistance marker (complement of nucleotides
563-1375).
[0152] Additional mutations are readily introduced into this
construct as desired, for example, to facilitate expression of
recombinant Phi 29 polymerases having modified active site regions.
See, e.g., SEQ ID NOs:15-23. The recombinant proteins can be
expressed in E. coli, for example, and purified using the GST, His,
and/or S tags and standard techniques. The tags are optionally
removed by digestion with an appropriate protease (e.g., thrombin
or enterokinase).
Example 2
Exemplary Recombinant Polymerases
[0153] A variety of recombinant Phi 29 polymerases with modified
active site regions have been constructed. Without intending to be
limited to any particular mechanism, the following examples
illustrate structural modifications that can reduce steric
inhibition for entry of nucleotide analogues into the modified
active site regions, coordinate extra phosphate groups by providing
features that complement these groups (e.g., positively charged
amino acid side chains), and/or otherwise enhance the ability of
the polymerase to incorporate nucleotide analogues.
[0154] FIG. 2 Panel A shows a sequence alignment of Phi 29-like
polymerases in the region surrounding residues 505-525, whose
position is indicated by the bracket. Amino acid residues differing
from Phi 29 are underlined. The majority of this domain is missing
in the cp-1 DNA polymerase (which, like G1, is more distantly
related to Phi 29). In addition, there is notably less sequence
conservation within the domain than in the flanking sequence. These
observations suggest that removal of the domain is unlikely to be
deleterious.
[0155] The top three views in FIG. 2 Panel B illustrate the
structure of the Phi 29 polymerase (see, e.g., Kamtekar et al.
(2004) "Insights into strand displacement and processivity from the
crystal structure of the protein-primed DNA polymerase of
bacteriophage .PHI.29" Mol. Cell. 16(4): 609-618). The bottom three
views show the polymerase with residues 505-525 removed,
illustrating that removal of this domain opens up the nucleotide
binding pocket. See, e.g., SEQ ID NOs:12 and 13 or 33 and 34, which
remove this domain using different turns.
[0156] FIG. 3 Panel A shows a sequence alignment of Phi 29-like
polymerases in the region surrounding E375 of Phi 29. The top three
views in Panel B illustrate the structure of the Phi 29 polymerase.
The glutamate at position 375 (indicated by the arrow) is located
proximal to the positively charged residues (K371, K379, K383;
depicted in green with blue knobs) that contact the triphosphate
moiety of the incoming dNTP. As illustrated in the bottom three
views in Panel B, this negatively charged amino acid (E) was
replaced with a positive one (H) in an attempt to coordinate the
extra phosphate in the tetraphosphate nucleotide analogues.
Additionally, the extra positive charge at this site may help
coordinate triphosphate analogs. Analysis of the recombinant
polymerase suggests that the E375H mutation has improved the
kinetics of the enzyme for incorporating phosphate labeled
nucleotide analogues (see Example 3 below). Mutant E375S was also
constructed to introduce a neutral residue at this location and/or,
e.g., to facilitate conformational change to enable function. See
also SEQ ID NOs:4-7 and 25-28.
[0157] FIG. 4 Panel A shows a sequence alignment of Phi 29-like
polymerases in the region surrounding E486 of Phi 29. The top three
views in Panel B illustrate the structure of the Phi 29 polymerase;
the location of E486 is indicated by an arrow. As illustrated in
the bottom three views, replacement of E486 by an alanine residue
creates more room in the active site region near the catalytic
carboxylates (D249 and D458, depicted in white) and removes a
negative charge. As another example, replacement of E486 by an
aspartic acid residue removes a carbon, decreasing steric
interference with nucleotide analogue binding while retaining the
negative charge. See also SEQ ID NOs:9-10 and 30-31.
[0158] FIG. 5 Panel A shows a sequence alignment of Phi 29-like
polymerases in the region surrounding K512 of Phi 29. The top three
views in Panel B illustrate the structure of the Phi 29 polymerase.
K512 (indicated by an arrow) juts out from the residue 505-525
domain and partially blocks the opening to the incoming dNTP
binding site. As illustrated in the bottom three views, replacement
of K512 by an alanine residue reduces steric inhibition for entry
of nucleotide analogues into the active site region, providing more
space for them to get into the binding pocket. See also SEQ ID
NOs:11 and 32.
[0159] FIG. 6 Panel A shows a sequence alignment of Phi 29-like
polymerases in the region surrounding K135 of Phi 29. The top three
views in Panel B illustrate the structure of the Phi 29 polymerase.
K135 (indicated by an arrow) juts into the opening to the incoming
dNTP binding site. As illustrated in the bottom three views,
replacement of K135 by an alanine residue reduces steric inhibition
for entry of nucleotide analogues into the active site region,
providing more space for them to get into the binding pocket. See
also SEQ ID NOs:3 and 24.
Example 3
Screening and Characterization of Recombinant Polymerases
[0160] Recombinant polymerases generated as in Example 2, or
through essentially any other rational or random mutagenesis
strategy, are optionally characterized to determine their
properties for various natural and/or nucleotides. One exemplary
five-step protocol for characterizing recombinant polymerases
follows.
[0161] The recombinant polymerase is initially evaluated on the
quality of the protein preparation and basic catalytic activity.
The polymerase's activity is analyzed with natural (native)
nucleotides, and its specific activity (units/mg) is determined.
Only catalytically competent mutants are selected for the next
steps.
[0162] The processivity (dissociation/kb) of the polymerase is
estimated in a primer extension reaction performed in the presence
of "Trap" (unlabeled competitor DNA or heparin). The processivity
assay is designed to select mutants that retain the capability to
synthesize a long DNA product in a continuous polymerization run
(without polymerization reinitiation) with natural nucleotides.
Mutants with a significant decrease in processivity are not
selected for the next step.
[0163] Polymerization rate (bases/min) with four analogues at 10
.mu.M (A488dA4P, A633dC4P, A546dG4P and A594dT4P) and circular
template (AGTC, a 72mer circular template largely consisting of a
repeating AGTC motif) is determined.
[0164] The most promising polymerase mutants are characterized by
determination of the polymerization rate and Km for A488dC4P and
A568dC4P and a subset of natural nucleotides (dATP, dGTP and dTTP),
using a circular template (AGTC). Velocity is measured at several
different concentrations of the analogs, A488dC4P (a representative
good substrate) and A568dC4P (a representative less preferred
substrate).
[0165] An initial selection for polymerase mutants with improved
kinetics for terminal phosphate labeled nucleotide analogues is
performed, using a primer extension assay with nucleotide analogues
to determine rate with analogues under experimental conditions. Two
separate experiments are typically performed, one in the presence
of 10 .mu.M A488dC4P, 20 .mu.M 3dNTPs-dCTP, and circular template
(AGTC), and one in the presence of 10 .mu.M A568dC4P, 20 .mu.M
3dNTPs-dCTP, and circular template (AGTC).
[0166] Other characteristics of the recombinant polymerase are
optionally examined, including, for example, fidelity, residence
time (1/V.sub.max), exonuclease activity (e.g., at 10 uM, via
extension of mismatched primer), active fraction (burst frequency),
rate with dNTPs, dN5Ps, linker-only analogs, and/or FRET analogs,
kinetics (ability to utilize analogs) with Mg.sup.2+ vs. Mn.sup.2+,
sensitivity to photodamage, single-stranded DNA binding, monomeric
state (e.g., using gel filtration or native gels), and/or
shelf-life.
[0167] Results of protein quality evaluation and polymerization
rate and kinetic constant determination for exemplary recombinant
Phi 29 polymerases are presented in Tables 1 and 2,
respectively.
TABLE-US-00002 TABLE 1 Initial characterization. Concentration;
Yield of Purified Specific Activity Description Polymerase
(units/mg) His-K135A-N62D 3.7 uM; 1 mg 12,454,000 His-E375H-N62D
7.4 uM; 1 mg 10,945,000 His-E375S-N62D 109 uM; 7 mg 10,961,000
His-E486A-N62D 40 uM; 3.5 mg 4,133,000 His-E486D-N62D 36 uM; 3.1 mg
11,634,000 His-K512A-N62D 34 uM; 10 mg 16,073,000
His-NipTuck_1-N62D 32 uM; 2.5 mg 12,400,000 His-NipTuck_2-N62D 4.4
uM; 0.3 mg 7,960,000
TABLE-US-00003 TABLE 2 Characterization of polymerization rate with
natural and analogue nucleotides. A B C D E F G H I J GST-N62D 780
1200 20 1660 74 346 236 65 0.9799 His-N62D 750 1020 21 391 237 68
0.9754 His-K135A- 840 880 24 292 154 43 0.9801 N62D His-E375H- 780
950 8 930 11 411 366 123 0.9510 N62D His-E375S- 940 1190 15 1300 28
420 332 74 0.9815 N62D His-E486A- 1690 303 118 15 0.9875 N62D
His-E486D- 220 134 15 0.9885 N62D His-K512A- 1590 359 196 34 0.9821
N62D (630) His- 660 520 24 153 116 24 0.9585 NipTuck_1- N62D His-
540 147 129 28 0.9520 NipTuck_2- (1840) N62D Column A: Description.
Column B: dTTP, dATP, dGTP (no G fork) V at 20 .mu.M; determined by
an assay with three native nucleotides (dGTP, dTTP and dATP).
Column C: A488dC4P, k.sub.el, (bp/min); determined by examining the
nucleotide analogue concentration dependence of the polymerization
rate. Column D: A488dC4P, Km; determined by examining the
nucleotide analogue concentration dependence of the polymerization
rate. Column E: A568dC4P, k.sub.el ; determined by examining the
nucleotide analogue concentration dependence of the polymerization
rate. Column F: A568dC4P, Km; determined by examining the
nucleotide analogue concentration dependence of the polymerization
rate. Column G: A488dC4P, V at 10 .mu.M; determined by an assay
with a single analogue at low concentration (10 uM) and three
native nucleotides. Column H: A568dC4P, V at 10 .mu.M; determined
by an assay with a single analogue at low concentration (10 uM) and
three native nucleotides. Column I: A488dA4P, A633dC4P, A546dG4P,
A594dT4P, V at 10 .mu.M; determined by an assay with four
terminally labeled nucleotide analogs. Column J: Processivity
(kb.sup.-1); determined by a processivity assay.
[0168] Assay with a Single Analogue at Low Concentration (10 .mu.M)
and Three Native Nucleotides
[0169] The .PHI.29 DNA polymerase (parental enzyme or mutant) was
preincubated with DNA template (72 nucleotide circular DNA
including mostly repetitive sequence AGTC) with annealed DNA
primer. The preincubation mix includes three native nucleotides
(dTTP, dATP and dGTP) and a terminal labeled nucleotide analogue
(A488dC4P or A568dC4P) at 10 .mu.M concentration. After a short
preincubation, the reaction was started with MnCl.sub.2. The
reaction was stopped with EDTA, and the products were separated
using agarose gel electrophoresis and stained with SYBR Gold
(Invitrogen). The average length of the DNA generated with DNA
polymerase was determined and used to estimate the polymerization
rate. See, e.g., Table 2 Columns G and H.
[0170] Assay with Four Terminally Labeled Nucleotide Analogs
[0171] The procedure is basically as described above in the section
entitled "Assay with a single analogue at low concentration (10
.mu.M) and three native nucleotides," with the exception that in
this assay all nucleotides are terminally labeled (A488dA4P,
A633dC4P, A546dG4P, A594dT4P all at 10 .mu.M). See, e.g., Table 2
Column I.
[0172] Assay with Three Native Nucleotides (dGTP, dTTP and
dATP)
[0173] The .PHI.29 DNA polymerase (parental enzyme or mutant) was
preincubated with DNA template (circular DNA including mostly
repetitive sequence CAT, no G residues) with annealed DNA primer;
the preincubation mix includes three native nucleotides (dTTP, dATP
and dGTP). All subsequent steps were basically as described above
in the section entitled "Assay with a single analogue at low
concentration (10 .mu.M) and three native nucleotides." See, e.g.,
Table 2 Column B.
[0174] The Nucleotide Analogue Concentration Dependence of the
Polymerization Rate
[0175] The .PHI.29 DNA polymerase (parental enzyme or mutant) was
preincubated with a DNA template (72 nucleotide circular DNA
including mostly repetitive sequence AGTC) with annealed DNA
primer. The preincubation mix includes also three native
nucleotides (dTTP, dATP and dGTP 20 .mu.M each) and various
concentrations of the terminally labeled analogue (A488dC4P or
A568dC4P). All subsequent steps were basically as described above
in the section entitled "Assay with a single analogue at low
concentration (10 .mu.M) and three native nucleotides." An average
length of the DNA products generated with DNA polymerase at an
individual analogue concentration was determined, and the results
were fitted with the equation k=k.sub.el*[S]*(K.sub.d+[S]).sup.-1
where k is the observed polymerization rate, k.sub.el is the
polymerization rate at saturating substrate concentration (k.sub.el
measures incorporation of multiple residues), and [S] is substrate
concentration. See, e.g., Table 2 Columns C, D, E, and F.
[0176] Processivity Assay
[0177] The .PHI.29 DNA polymerase (parental enzyme or mutant) was
preincubated with DNA template (72 nucleotide circular DNA
including mostly repetitive sequence AGTC) with annealed DNA
primer. After a short preincubation, the reaction was started with
a starting mix including MnCl.sub.2, dNTP and heparin. Including
the heparin in the reaction prevents polymerization from
reinitiating after the polymerase dissociates from the
template/primer, so that all generated DNA products are a result of
continuous polymerization runs. After 20 min incubation, the
reaction was stopped with EDTA and the products were separated
using agarose gel electrophoresis and stained with SYBR Gold
(Invitrogen). The DNA products were analyzed basically as described
in Bibillo A, Eickbush T H. J Biol Chem. 2002 Sep. 20;
277(38):34836-45, Epub 2002 Jul. 5. The results were fitted with
single exponential equation A*exp(-P.sub.off*kb) where A is
amplitude, P.sub.off is the probability of premature polymerase
dissociation, and kb is DNA length (1000 nucleotides). The
probability of chain elongation (processivity) can be readily
calculated by subtracting the P.sub.off value from 1.0. See, e.g.,
Table 2 Column J.
[0178] Sequences of Exemplary Recombinant Polymerases
[0179] Amino acid and polynucleotide sequences of wild-type Phi 29
and exemplary recombinant polymerases are presented in Table 3.
TABLE-US-00004 TABLE 3 Sequences. SEQ ID NO: Notes Sequence 1
wild-type mkhmprkmys cdfetttkve dcrvwaygym niedhseyki gnsldefmaw
Phi 29 vlkvqadlyf hnlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw amino
acid ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt vlkgdidyhk
sequence erpvgykitp eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd
iittkkfkkv fptlslgldk evryayrggf twlndrfkek eigegmvfdv nslypaqmys
rllpygepiv fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy kgneylkssg
geiadlwlsn vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi kttsegaikq
laklmlnsly gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg vfitawaryt
titaaqacyd riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk rakylrqkty
iqdiymkevd gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf srkmkpkpvq
vpggvvlvdd tftik 2 N62D amino mspilgywki kglvqptrll leyleekyee
hlyerdegdk wrnkkfelgl acid efpnlpyyid gdvkltqsma iiryiadkhn
mlggcpkera eismlegavl sequence dirygvsria yskdfetlkv dflsklpeml
kmfedrlchk tylngdhvth (tagged) pdfmlydald vvlymdpmcl dafpklvcfk
krieaipqid kylksskyia wplqgwqatf gggdhppksd gstsgsghhh hhhsaglvpr
gstaigmket aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh mprkmyscdf
etttkvedcr vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl kfdgafiinw
lerngfkwsa dglpntynti isrmgqwymi diclgykgkr kihtviydsl kklpfpvkki
akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii aealliqfkq gldrmtagsd
slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig egmvfdvnsl
ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq ikrsrfykgn
eylkssggei adlwlsnvd1 elmkehydly nveyisglkf kattglfkdf idkwtyiktt
segaikqlak lmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd pvytpmgvfi
tawaryttit aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw ahestfkrak
ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm tdkikkevtf enfkvgfsrk
mkpkpvqvpg gvvlvddtft ik 3 K135A- mspilgywki kglvqptrll leyleekyee
hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma iiryiadkhn
mlggcpkera eismlegavl acid dirygvsria yskdfetlkv dflsklpeml
kmfedrlchk tyingdhvth sequence pdfmlydald vvlymdpmcl dafpklvcfk
krieaipqid kylksskyia (tagged) wplqgwqatf gggdhppksd gstsgsghhh
hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh
mprkmyscdf etttkvedcr vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl
kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi diclgykgkr kihtviydsl
kklpfpvkki aadfkltvlk gdidyhkerp vgykitpeey ayikndiqii aealliqfkq
gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig
egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq
ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly nveyisglkf kattglfkdf
idkwtyiktt segaikqlak lmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd
pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw
ahestfkrak ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm tdkikkevtf
enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 4 E375H- mspilgywki kglvqptrll
leyleekyee hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma
iiryiadkhn mlggcpkera eismlegavl acid dirygvsria yskdfetlkv
dflsklpeml kmfedrlchk tylngdhvth sequence pdfmlydald vvlymdpmcl
dafpklvcfk krieaipqid kylksskyia (tagged) wplqgwqatf gggdhppksd
gstsgsghhh hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg sgddddkspm
gyrgsefmkh mprkmyscdf etttkvedcr vwaygymnie dhseykigns ldefmawvlk
vqadlyfhdl kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi diclgykgkr
kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii
aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl
ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe
lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly nveyisglkf
kattglfkdf idkwtyiktt shgaikqlak lmlnslygkf asnpdvtgkv pylkengalg
frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt gteipdvikd
ivdpkklgyw ahestfkrak ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm
tdkikkevtf enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 5 E375S- mspilgywki
kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid
gdvkltqsma iiryiadkhn mlggcpkera eismlegavl acid dirygvsria
yskdfetlkv dflsklpeml kmfedrlchk tylngdhvth sequence pdfmlydald
vvlymdpmcl dafpklvcfk krieaipqid kylksskyia (tagged) wplqgwqatf
gggdhppksd gstsgsghhh hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg
sgddddkspm gyrgsefmkh mprkmyscdf etttkvedcr vwaygymnie dhseykigns
ldefmawvlk vqadlyfhdl kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi
diclgykgkr kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey
ayikndiqii aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr
yayrggftwl ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl
hiqhircefe lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly
nveyisglkf kattglfkdf idkwtyiktt ssgaikqlak lmlnslygkf asnpdvtgkv
pylkengalg frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt
gteipdvikd ivdpkklgyw ahestfkrak ylrqktyiqd iymkevdgkl vegspddytd
ikfsvkcagm tdkikkevtf enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 6 E375K-
mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl N62D amino
efpnlpyyid gdvkltqsma iiryiadkhn mlggcpkera eismlegavl acid
dirygvsria yskdfetlkv dflsklpeml kmfedrlchk tylngdhvth sequence
pdfmlydald vvlymdpmcl dafpklvcfk krieaipqid kylksskyia (tagged)
wplqgwqatf gggdhppksd gstsgsghhh hhhsaglvpr gstaigmket aaakferqhm
dspdlgtggg sgddddkspm gyrgsefmkh mprkmyscdf etttkvedcr vwaygymnie
dhseykigns ldefmawvlk vqadlyfhdl kfdgafiinw lerngfkwsa dglpntynti
isrmgqwymi diclgykgkr kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp
vgykitpeey ayikndiqii aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt
lslgldkevr yayrggftwl ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg
kyvwdedypl hiqhircefe lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl
elmkehydly nveyisglkf kattglfkdf idkwtyiktt skgaikqlak lmlnslygkf
asnpdvtgkv pylkengalg frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii
ycdtdsihlt gteipdvikd ivdpkklgyw ahestfkrak ylrqktyiqd iymkevdgkl
vegspddytd ikfsvkcagm tdkikkevtf enfkvgfsrk mkpkpvqvpg gvvlvddtft
ik 7 E375R- mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl
N62D amino efpnlpyyid gdvkltqsma iiryiadkhn mlggcpkera eismlegavl
acid dirygvsria yskdfetlkv dflsklpeml kmfedrlchk tylngdhvth
sequence pdfmlydald vvlymdpmcl dafpklvcfk krieaipqid kylksskyia
(tagged) wplqgwqatf gggdhppksd gstsgsghhh hhhsaglvpr gstaigmket
aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh mprkmyscdf etttkvedcr
vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl kfdgafiinw lerngfkwsa
dglpntynti isrmgqwymi diclgykgkr kihtviydsl kklpfpvkki akdfkltvlk
gdidyhkerp vgykitpeey ayikndiqii aealliqfkq gldrmtagsd slkgfkdiit
tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig egmvfdvnsl ypaqmysrll
pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq ikrsrfykgn eylkssggei
adlwlsnvdl elmkehydly nveyisglkf kattglfkdf idkwtyiktt srgaikqlak
lmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd pvytpmgvfi tawaryttit
aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw ahestfkrak ylrqktyiqd
iymkevdgkl vegspddytd ikfsvkcagm tdkikkevtf enfkvgfsrk mkpkpvqvpg
gvvlvddtft ik 8 L384R- mspilgywki kglvqptrll leyleekyee hlyerdegdk
wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma iiryiadkhn mlggcpkera
eismlegavl acid dirygvsria yskdfetlkv dflsklpeml kmfedrlchk
tylngdhvth sequence pdfmlydald vvlymdpmcl dafpklvcfk krieaipqid
kylksskyia (tagged) wplqgwqatf gggdhppksd gstsgsghhh hhhsaglvpr
gstaigmket aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh mprkmyscdf
etttkvedcr vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl kfdgafiinw
lerngfkwsa dglpntynti isrmgqwymi diclgykgkr kihtviydsl kklpfpvkki
akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii aealliqfkq gldrmtagsd
slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig egmvfdvnsl
ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq ikrsrfykgn
eylkssggei adlwlsnvdl elmkehydly nveyisglkf kattglfkdf idkwtyiktt
segaikqlak rmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd pvytpmgvfi
tawaryttit aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw ahestfkrak
ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm tdkikkevtf enfkvgfsrk
mkpkpvqvpg gvvlvddtft ik 9 E486A- mspilgywki kglvqptrll leyleekyee
hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma iiryiadkhn
mlggcpkera eismlegavl acid dirygvsria yskdfetlkv dflsklpeml
kmfedrlchk tylngdhvth sequence pdfmlydald vvlymdpmcl dafpklvcfk
krieaipqid kylksskyia (tagged) wplqgwqatf gggdhppksd gstsgsghhh
hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh
mprkmyscdf etttkvedcr vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl
kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi diclgykgkr kihtviydsl
kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii aealliqfkq
gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig
egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq
ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly nveyisglkf kattglfkdf
idkwtyiktt segaikqlak lmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd
pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw
ahastfkrak ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm tdkikkevtf
enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 10 E486D- mspilgywki kglvqptrll
leyleekyee hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma
iiryiadkhn mlggcpkera eismlegavl acid dirygvsria yskdfetlkv
dflsklpeml kmfedrlchk tylngdhvth sequence pdfmlydald vvlymdpmcl
dafpklvcfk krieaipqid kylksskyia (tagged) wplqgwqatf gggdhppksd
gstsgsghhh hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg sgddddkspm
gyrgsefmkh mprkmyscdf etttkvedcr vwaygymnie dhseykigns ldefmawvlk
vqadlyfhdl kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi diclgykgkr
kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii
aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl
ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe
lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly nveyisglkf
kattglfkdf idkwtyiktt segaikqlak lmlnslygkf asnpdvtgkv pylkengalg
frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt gteipdvikd
ivdpkklgyw andstfkrak ylrqktyiqd iymkevdgkl vegspddytd ikfsvkcagm
tdkikkevtf enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 11 K512A- mspilgywki
kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid
gdvkltqsma iiryiadkhn mlggcpkera eismlegavl acid dirygvsria
yskdfetlkv dflsklpeml kmfedrlchk tylngdhvth sequence pdfmlydald
vvlymdpmcl dafpklvcfk krieaipqid kylksskyia (tagged) wplqgwqatf
gggdhppksd gstsgsghhh hhhsaglvpr gstaigmket aaakferqhm dspdlgtggg
sgddddkspm gyrgsefmkh mprkmyscdf etttkvedcr vwaygymnie dhseykigns
ldefmawvlk vqadlyfhdl kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi
diclgykgkr kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey
ayikndiqii aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr
yayrggftwl ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl
hiqhircefe lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly
nveyisglkf kattglfkdf idkwtyiktt segaikqlak lmlnslygkf asnpdvtgkv
pylkengalg frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt
gteipdvikd ivdpkklgyw ahestfkrak ylrqktyiqd iymkevdgal vegspddytd
ikfsvkcagm tdkikkevtf enfkvgfsrk mkpkpvqvpg gvvlvddtft ik 12
NipTuck_1- mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl
N62D amino efpnlpyyid gdvkltqsma iiryiadkhn mlggcpkera eismlegavl
acid dirygvsria yskdfetlkv dflsklpeml kmfedrlchk tylngdhvth
sequence pdfmlydald vvlymdpmcl dafpklvcfk krieaipqid kylksskyia
(deletion wplqgwqatf gggdhppksd gstsgsghhh hhhsaglvpr gstaigmket of
residues aaakferqhm dspdlgtggg sgddddkspm gyrgsefmkh mprkmyscdf
505-525) etttkvedcr vwaygymnie dhseykigns ldefmawvlk vqadlyfhdl
(tagged) kfdgafiinw lerngfkwsa dglpntynti isrmgqwymi diclgykgkr
kihtviydsl kklpfpvkki akdfkltvlk gdidyhkerp vgykitpeey ayikndiqii
aealliqfkq gldrmtagsd slkgfkdiit tkkfkkvfpt lslgldkevr yayrggftwl
ndrfkekeig egmvfdvnsl ypaqmysrll pygepivfeg kyvwdedypl hiqhircefe
lkegyiptiq ikrsrfykgn eylkssggei adlwlsnvdl elmkehydly nveyisglkf
kattglfkdf idkwtyiktt segaikqlak lmlnslygkf asnpdvtgkv pylkengalg
frlgeeetkd pvytpmgvfi tawaryttit aaqacydrii ycdtdsihlt gteipdvikd
ivdpkklgyw ahestfkrak ylrqktyiqd ikdgefsvkc agmtdkikke vtfenfkvgf
srkmkpkpvq vpggvvlvdd tftik 13 NipTuck_2- mspilgywki kglvqptrll
leyleekyee hlyerdegdk wrnkkfelgl N62D amino efpnlpyyid gdvkltqsma
iiryiadkhn mlggcpkera eismlegavl acid dirygvsria yskdfetlkv
dflsklpeml kmfedrlchk tylngdhvth sequence pdfmlydald vvlymdpmcl
dafpklvcfk krieaipqid kylksskyia (deletion wplqgwqatf gggdhppksd
gstsgsghhh hhhsaglvpr gstaigmket of residues aaakferqhm dspdlgtggg
sgddddkspm gyrgsefmkh mprkmyscdf 505-525) etttkvedcr vwaygymnie
dhseykigns ldefmawvlk vqadlyfhdl (tagged) kfdgafiinw lerngfkwsa
dglpntynti isrmgqwymi diclgykgkr kihtviydsl kklpfpvkki akdfkltvlk
gdidyhkerp vgykitpeey ayikndiqii aealliqfkq gldrmtagsd slkgfkdiit
tkkfkkvfpt lslgldkevr yayrggftwl ndrfkekeig egmvfdvnsl ypaqmysrll
pygepivfeg kyvwdedypl hiqhircefe lkegyiptiq ikrsrfykgn eylkssggei
adlwlsnvdl elmkehydly nveyisglkf kattglfkdf idkwtyiktt segaikqlak
lmlnslygkf asnpdvtgkv pylkengalg frlgeeetkd pvytpmgvfi tawaryttit
aaqacydrii ycdtdsihlt gteipdvikd ivdpkklgyw ahestfkrak ylrqktyiqd
idgfsvkcag mtdkikkevt fenfkvgfsr kmkpkpvqvp ggvvlvddtf tik 14 N62D
tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtgg nucleotide
tggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgct sequence-
cctttcgctttcttcccttcctttctcgccacgttcgccggctttccccg pET41
tcaagctctaaatcgggggctccctttagggttccgatttagtgctttac
N62D 1 ggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggg plasmid
ccatcgccctgatagacggtttttcgccctttgacgttggagtccacgtt
ctttaatagtggactcttgttccaaactggaacaacactcaaccctatct
cggtctattcttttgatttataagggattttgccgatttcggcctattgg
ttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaat
attaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaa
cccctatttgtttatttttctaaatacattcaaatatgtatccgctcatg
aattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttat
tcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaat
gaaggagaaaactcaccgaggcagttccataggatggcaagatcctggta
tcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcc
cctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgact
gaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttc
aacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaac
cgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctg
ttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacac
tgccagcgcatcaacaatattttcacctgaatcaggatattcttctaata
cctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatca
tcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgt
cagccagtttagtctgaccatctcatctgtaacatcattggcaacgctac
ctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaat
cgatagattgtcgcacctgattgcccgacattatcgcgagcccatttata
cccatataaatcagcatccatgttggaatttaatcgcggcctagagcaag
acgtttcccgttgaatatggctcataacaccccttgtattactgtttatg
taagcagacagttttattgttcatgaccaaaatcccttaacgtgagtttt
cgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttga
gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccacc
gctaccagcggtggtttgtttgccggatcaagagctaccaactctttttc
cgaaggtaactggcttcagcagagcgcagataccaaatactgtccttcta
gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctac
atacctcgctctgctaatcctgttaccagtggctgctgccagtggcgata
agtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcg
cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg
aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcg
ccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagg
gtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggta
tctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttt
tgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg
gcctttttacggttcctggccttttgctggccttttgctcacatgttctt
tcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagt
gagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtg
agcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatct
gtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctc
tgatgccgcatagttaagccagtatacactccgctatcgctacgtgactg
ggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgac
gggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctcc
gggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgag
gcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgt
ctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaat
gtctggcttctgataaagcgggccatgttaagggcggttttttcctgttt
ggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatga
taccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaa
catgcccggttactggaacgttgtgagggtaaacaactggcggtatggat
gcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgtta
atacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcag
atccggaacataatggtgcagggcgctgacttccgcgtttccagacttta
cgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacg
ttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattc
tgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacag
gagcacgatcatgctagtcatgccccgcgcccaccggaaggagctgactg
ggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtg
agctaacttacattaattgcgttgcgctcactgcccgctttccagtcggg
aaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagag
gcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagac
gggcaacagctgattgcccttcaccgcctggccctgagagagttgcagca
agcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtg
gttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccac
taccgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgca
ttgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacg
atgccctcattcagcatttgcatggtttgttgaaaaccggacatggcact
ccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagat
atttatgccagccagccagacgcagacgcgccgagacagaacttaatggg
cccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccac
gcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtg
tctggtcagagacatcaagaaataacgccggaacattagtgcaggcagct
tccacagcaatggcatcctggtcatccagcggatagttaatgatcagccc
actgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcga
cgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcg
gcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccag
actggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgtt
gtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccact
ttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcggga
aacggtctgataagagacaccggcatactctgcgacatcgtataacgtta
ctggtttcacattcaccaccctgaattgactctcttccgggcgctatcat
gccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgac
gctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttg
aggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggc
gcccaacagtcccccggccacggggcctgccaccatacccacgccgaaac
aagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatg
tcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggc
cacgatgcgtccggcgtagaggatcgagatcgatctcgatcccgcgaaat
taatacgactcactataggggaattgtgagcggataacaattcccctcta
gaaataattttgtttaactttaagaaggagatatacatatgtcccctata
ctaggttattggaaaattaagggccttgtgcaacccactcgacttctttt
ggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaag
gtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaat
cttccttattatattgatggtgatgttaaattaacacagtctatggccat
catacgttatatagctgacaagcacaacatgttgggtggttgtccaaaag
agcgtgcagagatttcaatgcttgaaggagcggttttggatattagatac
ggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttga
ttttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttat
gtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatg
ttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctgga
tgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccac
aaattgataagtacttgaaatccagcaagtatatagcatggcctttgcag
ggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatgg
ttcaactagtggttctggtcatcaccatcaccatcactccgcgggtctgg
tgccacgcggtagtactgcaattggtatgaaagaaaccgctgctgctaaa
ttcgaacgccagcacatggacagcccagatctgggtaccggtggtggctc
cggtgatgacgacgacaagagtcccatgggatatcggggatccgaattca
tgaagcatatgccgagaaagatgtatagttgtgactttgagacaactact
aaagtggaagactgtagggtatgggcgtatggttatatgaatatagaaga
tcacagtgagtacaaaataggtaatagcctggatgagtttatggcgtggg
tgttgaaggtacaagctgatctatatttccatgatctcaaatttgacgga
gcttttatcattaactggttggaacgtaatggttttaagtggtcggctga
cggattgccaaacacatataatacgatcatatctcgcatgggacaatggt
acatgattgatatatgtttaggctacaaagggaaacgtaagatacataca
gtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagc
taaagactttaaactaactgttcttaaaggtgatattgattaccacaaag
aaagaccagtcggctataagataacacccgaagaatacgcctatattaaa
aacgatattcagattattgcggaagctctgttaattcagtttaagcaagg
tttagaccggatgacagcaggcagtgacagtctaaaaggtttcaaggata
ttataaccactaagaaattcaaaaaggtgtttcctacattgagtcttgga
ctcgataaggaagtgagatacgcctatagaggtggttttacatggttaaa
tgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgtta
atagtctatatcctgcacagatgtatagtcgtctccttccatatggtgaa
cctatagtattcgagggtaaatacgtttgggacgaagattacccactaca
catacagcatatcagatgtgagttcgaattgaaagagggctatataccca
ctatacagataaaaagaagtaggttttataaaggtaatgagtacctaaaa
agtagcggcggggagatagccgacctctggttgtcaaatgtagacctaga
attaatgaaagaacactacgatttatataacgttgaatatatcagcggct
taaaatttaaagcaactacaggtttgtttaaagattttatagataaatgg
acgtacatcaagacgacatcagaaggagcgatcaagcaactagcaaaact
gatgttaaacagtctatacggtaaattcgctagtaaccctgatgttacag
ggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttgga
gaagaggaaacaaaagaccctgtttatacacctatgggcgttttcatcac
tgcatgggctagatacacgacaattacagcggcacaggcttgttatgatc
ggataatatactgtgatactgacagcatacatttaacgggtacagagata
cctgatgtaataaaagatatagttgaccctaagaaattgggatactgggc
acatgaaagtacattcaaaagagctaaatatctgagacagaagacctata
tacaagacatctatatgaaagaagtagatggtaagttagtagaaggtagt
ccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgac
tgacaagattaagaaagaggttacgtttgagaatttcaaagtcggattca
gtcggaaaatgaagcctaagcctgtgcaagtgccgggcggggtggttctg
gttgatgacacattcacaatcaaataagaattctgtacaggccttggcgc
gcctgcaggcgagctccgtcgacaagcttgcggccgcactcgagcaccac
caccaccaccaccaccactaattgattaatacctaggctgctaaacaaag
cccgaaaggaagctgagttggctgctgccaccgctgagcaataactagca
taaccccttggggcctctaaacgggtcttgaggggttttttgctgaaagg
aggaactatatccggat 15 K135A-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctgccgactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatacaaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 16 E375H-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cacacggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 17 E375S-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagcggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
caagcggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagctggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 18 L384R-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaacggatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 19 E486A-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgatcgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagacacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgccagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 20 E486D-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgacagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtaagttag
tagaaggtagtccagatgattacactgatataaatcttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 21 K512A-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagact-
gtaggg N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggcaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttacgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggacactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatctatatgaaagaagtagatggtgccttag
tagaaggtagtccagatgattacactgatataaaatttagtgttaaatgtgcgggaatgactgacaagat
taagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcctaagcctgtgcaa
gtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 22 NipTuck_1-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagactgtaggg
N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagataacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaatgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatcaaggatggagagtttagtgttaaatgtg
cgggaatgactgacaagattaagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaat
gaagcctaagcctgtgcaagtgccgggcggggtggttctggttgatgacacattcacaatcaaataa
23 NipTuck_2-
atgaagcacatgccgagaaagatgtatagttgtgactttgagacaactactaaagtggaagactgtaggg
N62D
tatgggcgtatggttatatgaatatagaagatcacagtgagtacaaaataggtaatagcctggatgag-
tt nucleotide
tatggcgtgggtgttgaaggtacaagctgatctatatttccatgatctcaaatttgacggagcttttatc
sequence
attaactggttggaacgtaatggttttaagtggtcggctgacggattgccaaacacatataata-
cgatca
tatctcgcatgggacaatggtacatgattgatatatgtttaggctacaaagggaaacgtaagatacatac
agtgatatatgacagcttaaagaaactaccgtttcctgttaagaagatagctaaagactttaaactaact
gttcttaaaggtgatattgattaccacaaagaaagaccagtcggctataagacaacacccgaagaatacg
cctatattaaaaacgatattcagattattgcggaagctctgttaattcagtttaagcaaggtttagaccg
gatgacagcaggcagtgacagtctaaaaggtttcaaggatattataaccactaagaaattcaaaaaggtg
tttcctacattgagtcttggactcgataaggaagtgagatacgcctatagaggtggttttacatggttaa
atgataggttcaaagaaaaagaaatcggagaaggcatggtcttcgatgttaatagtctatatcctgcaca
gatgtatagtcgtctccttccatacggtgaacctatagtattcgagggtaaatacgtttgggacgaagat
tacccactacacatacagcatatcagatgtgagttcgaattgaaagagggctatatacccactatacaga
taaaaagaagtaggttttataaaggtaatgagtacctaaaaagtagcggcggggagatagccgacctctg
gttgtcaaatgtagacctagaattaacgaaagaacactacgatttatataacgttgaatatatcagcggc
ttaaaatttaaagcaactacaggtttgtttaaagattttatagataaatggacgtacatcaagacgacat
cagaaggagcgatcaagcaactagcaaaactgatgttaaacagtctatacggtaaattcgctagtaaccc
tgatgttacagggaaagtcccttatttaaaagagaatggggcgctaggtttcagacttggagaagaggaa
acaaaagaccctgtttatacacctatgggcgttttcatcactgcatgggctagatacacgacaattacag
cggcacaggcttgttatgatcggataatatactgtgatactgacagcatacatttaacgggtacagagat
acctgatgtaataaaagatatagttgaccctaagaaattgggatactgggcacatgaaagtacattcaaa
agagctaaatatctgagacagaagacctatatacaagacatcgacggctttagtgttaaatgtgcgggaa
tgactgacaagattaagaaagaggttacgtttgagaatttcaaagtcggattcagtcggaaaatgaagcc
taagcctgtgcaagtgccgggcggggtggttctggttgatgacacattcacaatcaaataa 24
K135A- mkhmprkmys cdfetttkve dcrvwaygym niedhseyki gnsldefmaw N62D
amino vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw acid
ymidiclgyk gkrkihtviy dslkklpfpv kkiaadfklt vlkgdidyhk sequence
erpvgykitp eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv
fptlslgldk evryayrggf twlndrfkek eigegmvfdv nslypaqmys rllpygepiv
fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn
vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi kttsegaikq laklmlnsly
gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd
riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk rakylrqkty iqdiymkevd
gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd
tftik 25 E375H- mkhmprkmys cdfetttkve dcrvwaygym niedhseyki
gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty
ntiisrmgqw acid ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt
vlkgdidyhk sequence erpvgykitp eeyayikndi qiiaealliq fkqgldrmta
gsdslkgfkd iittkkfkkv fptlslgldk evryayrggf twlndrfkek eigegmvfdv
nslypaqmys rllpygepiv fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy
kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi
kttshgaikq laklmlnsly gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg
vfitawaryt titaaqacyd riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk
rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf
srkmkpkpvq vpggvvlvdd tftik 26 E375S- mkhmprkmys cdfetttkve
dcrvwaygym niedhseyki gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi
inwlerngfk wsadglpnty ntiisrmgqw acid ymidiclgyk gkrkihtviy
dslkklpfpv kkiakdfklt vlkgdidyhk sequence erpvgykitp eeyayikndi
qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv fptlslgldk evryayrggf
twlndrfkek eigegmvfdv nslypaqmys rllpygepiv fegkyvwded yplhiqhirc
efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg
lkfkattglf kdfidkwtyi kttssgaikq laklmlnsly gkfasnpdvt gkvpylkeng
algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd riiycdtdsi hltgteipdv
ikdivdpkkl gywahestfk rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc
agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd tftik 27 E375K-
mkhmprkmys cdfetttkve dcrvwaygym niedhseyki gnsldefmaw N62D amino
vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw acid
ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt vlkgdidyhk sequence
erpvgykitp eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv
fptlslgldk evryayrggf twlndrfkek eigegmvfdv nslypaqmys rllpygepiv
fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn
vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi kttskgaikq laklmlnsly
gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd
riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk rakylrqkty iqdiymkevd
gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd
tftik 28 E375R- mkhmprkmys cdfetttkve dcrvwaygym niedhseyki
gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty
ntiisrmgqw acid ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt
vlkgdidyhk sequence erpvgykitp eeyayikndi qiiaealliq fkqgldrmta
gsdslkgfkd iittkkfkkv fptlslgldk evryayrggf twlndrfkek eigegmvfdv
nslypaqmys rllpygepiv fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy
kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi
kttsrgaikq laklmlnsly gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg
vfitawaryt titaaqacyd riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk
rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf
srkmkpkpvq vpggvvlvdd tftik 29 L384R- mkhmprkmys cdfetttkve
dcrvwaygym niedhseyki gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi
inwlerngfk wsadglpnty ntiisrmgqw acid ymidiclgyk gkrkihtviy
dslkklpfpv kkiakdfklt vlkgdidyhk sequence erpvgykitp eeyayikndi
qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv fptlslgldk evryayrggf
twlndrfkek eigegmvfdv nslypaqmys rllpygepiv fegkyvwded yplhiqhirc
efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg
lkfkattglf kdfidkwtyi kttsegaikq lakrmlnsly gkfasnpdvt gkvpylkeng
algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd riiycdtdsi hltgteipdv
ikdivdpkkl gywahestfk rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc
agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd tftik 30 E486A-
mkhmprkmys cdfetttkve dcrvwaygym niedhseyki gnsldefmaw N62D amino
vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw acid
ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt vlkgdidyhk sequence
erpvgykitp eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv
fptlslgldk evryayrggf twlndrfkek eigegmvfdv
nslypaqmys rllpygepiv fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy
kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi
kttsegaikq laklmlnsly gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg
vfitawaryt titaaqacyd riiycdtdsi hltgteipdv ikdivdpkkl gywahastfk
rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf
srkmkpkpvq vpggvvlvdd tftik 31 E486D- mkhmprkmys cdfetttkve
dcrvwaygym niedhseyki gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi
inwlerngfk wsadglpnty ntiisrmgqw acid ymidiclgyk gkrkihtviy
dslkklpfpv kkiakdfklt vlkgdidyhk sequence erpvgykitp eeyayikndi
qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv fptlslgldk evryayrggf
twlndrfkek eigegmvfdv nslypaqmys rllpygepiv fegkyvwded yplhiqhirc
efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg
lkfkattglf kdfidkwtyi kttsegaikq laklmlnsly gkfasnpdvt gkvpylkeng
algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd riiycdtdsi hltgteipdv
ikdivdpkkl gywandstfk rakylrqkty iqdiymkevd gklvegspdd ytdikfsvkc
agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd tftik 32 K512A-
mkhmprkmys cdfetttkve dcrvwaygym niedhseyki gnsldefmaw N62D amino
vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw acid
ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt vlkgdidyhk sequence
erpvgykitp eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd iittkkfkkv
fptlslgldk evryayrggf twlndrfkek eigegmvfdv nslypaqmys rllpygepiv
fegkyvwded yplhiqhirc efelkegyip tiqikrsrfy kgneylkssg geiadlwlsn
vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi kttsegaikq laklmlnsly
gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd
riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk rakylrqkty iqdiymkevd
galvegspdd ytdikfsvkc agmtdkikke vtfenfkvgf srkmkpkpvq vpggvvlvdd
tftik 33 NipTuck_1- mkhmprkmys cdfetttkve dcrvwaygym niedhseyki
gnsldefmaw N62D amino vlkvqadlyf hdlkfdgafi inwlerngfk wsadglpnty
ntiisrmgqw acid ymidiclgyk gkrkihtviy dslkklpfpv kkiakdfklt
vlkgdidyhk sequence erpvgykitp eeyayikndi qiiaealliq fkqgldrmta
gsdslkgfkd (deletion iittkkfkkv fptlslgldk evryayrggf twlndrfkek
eigegmvfdv of residues nslypaqmys rllpygepiv fegkyvwded yplhiqhirc
efelkegyip 505-525) tiqikrsrfy kgneylkssg geiadlwlsn vdlelmkehy
dlynveyisg lkfkattglf kdfidkwtyi kttsegaikq laklmlnsly gkfasnpdvt
gkvpylkeng algfrlgeee tkdpvytpmg vfitawaryt titaaqacyd riiycdtdsi
hltgteipdv ikdivdpkkl gywahestfk rakylrqkty iqdikdgefs vkcaymtdki
kkevtfenfk vgfsrkmkpk pvqvpggvvl vddtftik 34 NipTuck_2- mkhmprkmys
cdfetttkve dcrvwaygym niedhseyki gnsldefmaw N62D amino vlkvqadlyf
hdlkfdgafi inwlerngfk wsadglpnty ntiisrmgqw acid ymidiclgyk
gkrkihtviy dslkklpfpv kkiakdfklt vlkgdidyhk sequence erpvgykitp
eeyayikndi qiiaealliq fkqgldrmta gsdslkgfkd (deletion iittkkfkkv
fptlslgldk evryayrggf twlndrfkek eigegmvfdv of residues nslypaqmys
rllpygepiv fegkyvwded yplhiqhirc efelkegyip 505-525) tiqikrsrfy
kgneylkssg geiadlwlsn vdlelmkehy dlynveyisg lkfkattglf kdfidkwtyi
kttsegaikq laklmlnsly gkfasnpdvt gkvpylkeng algfrlgeee tkdpvytpmg
vfitawaryt titaaqacyd riiycdtdsi hltgteipdv ikdivdpkkl gywahestfk
rakylrqkty iqdidgfsvk cagmtdkikk evtfenfkvg fsrkmkpkpv qvpggvvlvd
dtftik
[0180] Characterization of Recombinant Polymerases with Nucleotide
Analogues
[0181] K.sub.m and V.sub.max were determined for exemplary
recombinant Phi29 polymerases and various nucleotide analogues.
Results are presented in Table 4.
TABLE-US-00005 TABLE 4 K.sub.m and V.sub.max versus analogues.
Mutation Km.sup.1 Vmax.sup.1 Km.sup.2 Vmax.sup.2 Km.sup.3
Vmax.sup.3 Km.sup.4 Vmax.sup.4 N62D 23 610 20 540 838 2500 68 1620
N62D:E375H 17 800 15 526 433 1250 N62D:E375S 16.5 1158 40 1981
N62D:E375K 12 595 N62D:E375Y 2.5 773 6.6 471 440 1430 18 1292
N62D:E375W 1.8 889 5.0 595 248 1428 16 1585 .sup.1Measured for
Alexa633-O-dC4P (also referred to as A633dC4P herein)
.sup.2Measured for Alexa555-C2-dT4P. This analogue has a 2-carbon
linker ("C2") between the delta phosphate and the label moiety and
has the following structure: ##STR00005## .sup.3Measured for
Alexa555-C2-dTTP .sup.4Measured for Alexa532-O-dG4P
[0182] A set of exemplary recombinant Phi29 polymerases were
characterized with various nucleotides and/or nucleotide analogues.
Results are presented in Table 5.
TABLE-US-00006 TABLE 5 Screening data. Ratio Rate Hz Mutation
(Low/High).sup.1 High.sup.2 Rate.sup.3 Mutation 2.sup.4
Tag(s).sup.5 E375W 0.677 699.4 19.1 N62D His E375Y 0.694 498.5 12.1
N62D His E375H 0.445 510.1 9.4 N62D His E375Q 0.356 531.6 6.3 N62D
His E375K 0.425 516.1 6.2 N62D His E375S 0.335 528.4 5.9 N62D His
E375A 0.383 465.9 5.8 N62D His T15I 0.355 416.5 3.0 His N62D 0.355
349.3 2.8 GST-His N62D 0.362 373.2 2.7 His K135A 0.412 272.0 1.6
N62D His K512A 0.335 347.7 1.4 N62D His NipTuck1 0.508 192.4 1.3
N62D His D12A 0.888 55.3 1.2 GST-His E486A 0.441 152.5 1.0 N62D His
E486D 0.467 142.0 0.9 N62D His T15I 0.726 105.9 0.0 N62D His
NipTuck2 0.635 156.3 0.0 N62D His L384R 0.768 79.6 -0 N62D His
.sup.1Ratio = (rate at 5 .mu.M A633dC4P with 20 .mu.M dA, dG,
dTTP)/(rate at 25 .mu.M A633dC4P with 20 .mu.M dA, dG, dTTP) at 1
mM MnCl.sub.2. A higher ratio corresponds to a lower Km. .sup.2Rate
at 25 .mu.M A633dC4P with 20 .mu.M dA, dG, dTTP .sup.3Rate at 10
.mu.M Alexa488-O-dA4P, 10 .mu.M FAM-Alexa532-O-dG4P, 10 .mu.M
FAM-Alexa594-O-dT4P, 10 .mu.M Alexa633-O-dC4P with 1 mM MnCl.sub.2.
Provides a measure of both Km and Vmax, with a representative set
of four nucleotide analogues. .sup.4Background mutation (if any).
The recombinant polymerase corresponds to wild type Phi29
polymerase plus mutation 1 plus mutation 2. .sup.5Tag for
immobilization and or purification
[0183] Rates of binding and product release were determined for
exemplary recombinant Phi29 polymerases with nucleotide analogue
A594-dT4P using a FRET stopped flow assay as schematically
illustrated in FIG. 7 Panel A. Results are depicted graphically in
FIG. 7 for Phi29 N62D (Panel B), N62D:E375Y (Panel C), and
N62D:E375W (Panel D). Product release rates are shown in Table
6.
[0184] The E375Y and E375W mutant polymerases demonstrated
increased rates of binding and product release, indicating they
utilize the analogue better than does the parent enzyme.
TABLE-US-00007 TABLE 6 Product release rate Enzyme Product Release
Rate N62D 55 s.sup.-1 N62D:E375Y 117 s.sup.-1 N62D:E375W 76
s.sup.-1
[0185] Relative branching rate (dissociation of the analogue
without incorporation, i.e., substrate dissociation) was also
determined for exemplary recombinant Phi29 polymerases with
nucleotide analogue Alexa568-dA4P (also called A568-dA4P), using a
FRET stopped flow assay as schematically illustrated in FIG. 8
Panel A. In this technique, a template with a FRET donor dye
compatible for FRET with the corresponding dye on the nucleotide
analogue is employed. The primer has a dideoxy-termination at the
3' end to prevent incorporation. The analogue is pre-mixed with the
enzyme-template-dideoxyprimer complex. In the stopped flow
apparatus, this preformed complex is rapidly mixed with the
corresponding native nucleotide (native dATP, in this example) in
excess which serves as a "trap" to prevent rebinding of the
analogue after it dissociates. The increase in donor dye
fluorescence is monitored as a means of monitoring the
dissociation/branching rate of the analogue.
[0186] Results are depicted graphically in FIG. 8 for Phi29 N62D
(Panel B), N62D:E375Y (Panel C), and N62D:E375W (Panel D).
Branching rates are shown in Table 7.
TABLE-US-00008 TABLE 7 Branching rate. Enzyme Branching Rate N62D
90 s.sup.-1 N62D:E375Y 31 s.sup.-1 N62D:E375W 43 s.sup.-1
[0187] Additional Exemplary Recombinant Polymerases
[0188] Polymerases of the invention can include a Phi29 polymerase
(or homolog thereof) including any of the mutations listed in Table
8, singly or in combination with other mutations (e.g., other
mutations described herein). For example, polymerases of the
invention optionally include a Phi29 polymerase (or homolog
thereof) that includes a combination of mutations as specified in
Table 8.
TABLE-US-00009 TABLE 8 Exemplary mutations. D12A E375W T372D D12A
E375W T372E D12A E375W T372R K478D D12A E375W T372R K478E D12A
E375W T372K K478D D12A E375W T372K D478E D12A E375W K135D D12A
E375W K135E D12A E375W K512D D12A E375W K512E D12A E375W E408K D12A
E375W E408R D12A E375W T368D L480K D12A E375W T368E L480K D12A
D456N N62D D456N D12A D456A N62D D456A D12A D456S N62D D456S N62D
E375M N62D E375L N62D E375I N62D E375F N62D E375D D12A K512W N62D
K512W D12A K512Y N62D K512Y D12A K512F N62D K512F D12A E375W K512L
N62D E375W K512L D12A E375W K512Y N62D E375W K512Y D12A E375W K512F
N62D E375W K512F D12A E375Y K512L N62D E375Y K512L D12A E375Y K512Y
N62D E375Y K512Y D12A E375Y K512F N62D E375Y K512F D12A E375W K512H
N62D E375W K512H D12A E375Y K512H N62D E375Y K512H D12A D510F N62D
D510F D12A D510Y N62D D510Y D12A D510W N62D D510W D12A E375W D510F
N62D E375W D510F D12A E375W D510Y N62D E375W D510Y D12A E375W D510W
N62D E375W D510W D12A E375W D510W K512L N62D E375W D510W K512L D12A
E375W D510W K512F N62D E375W D510W K512F D12A E375W D510H N62D
E375W D510H D12A E375W D510H K512H N62D E375W D510H K512H D12A
E375W D510H K512F N62D E375W D510H K512F D12A V509Y N62D V509Y D12A
V509W N62D V509W D12A V509F N62D V509F D12A V514Y N62D V514Y D12A
V514W N62D V514W D12A V514F N62D V514F D12S D12N D12Q D12K D12A
N62D Y254F N62D Y254V N62D Y254A N62D Y390F N62D Y390A N62D S252A
N62D N387A N62D K157E N62D 1242H N62D Y259S N62D G320C N62D L328V
N62D T368M N62D T368G N62D Y369R N62D Y369H N62D Y369E N62D I370V
N62D I370K N62D K371Q N62D T372N N62D T372D N62D T372R N62D T372L
N62D T373A N62D T373H N62D S374E N62D I378K N62D K379E N62D K379T
N62D N387D N62D Y405V N62D L408D N62D G413D N62D D423V N62D I442V
N62D Y449F N62D D456V N62D L480M N62D V509K N62D V509I N62D D510A
N62D V514I N62D V514K N62D E515K N62D D523T N62D H149Y E375W M554S
M8S N62D M102S H116Y M188S E375W N62D M97S E375W M8S N62D M97S
M102S M188S E375W M554S M8A N62D M97A M102A M188A E375W M554A
[0189] A few mutations in the Phi29 polymerase have been previously
described. For the N62D mutation, see de Vega et al. (1996)
"Primer-terminus stabilization at the 3'-5' exonuclease active site
of phi29 DNA polymerase. Involvement of two amino acid residues
highly conserved in proofreading DNA polymerases" EMBO J.
15(5):1182-92. For the D12A mutation and mutations at positions
E14, 66, 165, 169, 12 and 66, and 14 and 66, see Esteban et al.
(1994) "3'-->5' exonuclease active site of phi 29 DNA
polymerase. Evidence favoring a metal ion-assisted reaction
mechanism" J Biol Chem. 269(50):31946-54. For mutation of S252, see
Blasco et al. (1993) "Phi 29 DNA polymerase active site. Residue
ASP249 of conserved amino acid motif `Dx2SLYP` is critical for
synthetic activities" J Biol Chem. 268(32):24106-13. For mutation
of Y254, see Blasco et al. (1992) "Phi 29 DNA polymerase active
site. Mutants in conserved residues Tyr254 and Tyr390 are affected
in dNTP binding" J Biol Chem. 267(27):19427-34. For mutation of
K371, see Truniger et al. (2002) "A positively charged residue of
phi29 DNA polymerase, highly conserved in DNA polymerases from
families A and B, is involved in binding the incoming nucleotide"
Nucleic Acids Res. 30(7):1483-92. For mutation of K379, see
Truniger et al. (2004) "Two Positively Charged Residues of .phi.29
DNA Polymerase, Conserved in Protein-primed DNA Polymerases, are
Involved in Stabilisation of the Incoming Nucleotide" Journal of
Molecular Biology 335(2):481-494. For mutation of N387, see Blasco
et al. (1993) "Phi 29 DNA polymerase active site. The conserved
amino acid motif `Kx3NSxYG` is involved in template-primer binding
and dNTP selection" J Biol Chem. 268(22):16763-70. For mutation of
Y390, see Blasco et al (1992) "Phi 29 DNA polymerase active site.
Mutants in conserved residues Tyr254 and Tyr390 are affected in
dNTP binding" J Biol Chem. 267(27):19427-34. For mutation of D456,
see Bernad et al. (1990) "The highly conserved amino acid sequence
motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is
required by phage phi 29 DNA polymerase for protein-primed
initiation and polymerization" Proc Natl Acad Sci USA.
87(12):4610-4.
Example 4
A Computational Framework for Modeling and Testing the Enzymatic
Kinetics of DNA Polymerase, Addressing all Kinetic Processes and
Free Variables Simultaneously
[0190] Polymerase kinetic state transitions are stored in a
probability matrix for discrete time steps. A vector of
probabilistic state distributions may describe the probability of
finding a particular polymerase in a number of polymerase states
according to a continuum model. Linear algebra multiplication of
the state distribution vector with the state transition probability
matrix gives a new vector of polymerase state distributions,
describing the effect of the passage of time equal to the discrete
time step of the state transition probability matrix.
[ template 1 template 2 ] * [ kinetic_matrix ] = [ new state
distributions ] ##EQU00001##
By raising the state transition probability matrix to a particular
exponential power (eg. 100), we simulate the passage of time of a
particular number of discrete time steps (eg. 100 time steps).
Using many discrete time steps we simulate DNA polymerization.
Steady State Model.
1000 { [ template 1 template 2 template 1000 ] 656 * [ 656 .times.
656 kinetic_matrix ] 100 = [ 1000 .times. 656 new state
distributions ] ##EQU00002##
The transition rates are user-defined. The probability matrix is
automatically generated using the template sequence and hard-coded
state transition rules. A variety of parameters, such as reagent
concentrations, kinetic rate values, and probability matrix
organization can vary from those described in this example.
[0191] The following is an example of a steady state polymerase
kinetic model.
R p = C 6 K 61 - C 1 K 16 = C 1 K 12 - C 2 K 21 = C 2 K 23 - C 3 K
32 = C 3 K 34 - C 4 K 43 = C 4 K 45 = C 5 K 56 - C 6 K 65
##EQU00003##
R.sub.p=rate of catalysis C.sub.6=probability of finding polymerase
in state 6 K.sub.61=transition rate of polymerase in state 6 to
state 1 k.sub.ij=reaction rated P.sub.ij=k.sub.ij.DELTA.t reaction
rated P.sub.ij=i.fwdarw.j probability *K.sub.54.apprxeq.0 as
concentration of pyrophosphate .dwnarw.
R.sub.p=C.sub.6K.sub.61-C.sub.1K.sub.16=rate of catalysis
R.sub.p=(R.sub.p).sub.max @ K.sub.61.fwdarw..infin.,
C.sub.6.fwdarw.0 as a condition of nucleotide concentration
increasing to saturation
[0192] Mega Matrix
[0193] The following is a single 2-D matrix to capture all possible
kinetic states of a polymerase-template-dNTP system:
TABLE-US-00010 Variables Previous Pol State Template Base Nucleo.
Base Native/Analog Nucleo. Base 1-4 A-T A-T 0-1 A-T 5 A-T A-T X A-T
6 A-T X X A-T 7 A-T A-T X A-T
[0194] *This results in a 656-state matrix, where the states are as
follows:
TABLE-US-00011 1. 1 A A 0 A 2. 1 A A 0 C 3. 1 A A 0 G 4. 1 A A 0 T
5. 1 A A 1 A 6. 1 A A 1 C 7. 1 A A 1 G 8. 1 A A 1 T 9. 1 A C 0 A
652. 7* T G X T 653. 7* T T X A 654. 7* T T X C 655. 7* T T X G
656. 7* T T X T *In this case the state 7 is dissociation of the
polymerase from the template, which may optionally be simplified to
never happen.
[0195] In this case the DNA template is the repeated sequence
(ACGT.) For a longer template repeated sequence there will be
proportionally more states, to the extent that the longer template
repeated sequence does not contain the original template sequence.
For example, the probability transition matrix generated for the
sequence
TABLE-US-00012 . . . [ACGT]ACGT . . .
would be equivalent to the matrix generated for the sequence
TABLE-US-00013 . . . [ACGTACGT]ACGT . . .
However, the probability transition matrix generated for the
sequence
TABLE-US-00014 . . . [AACCGGTT]AACC . . .
would be different, as it contains many state transitions not
allowed in the original matrix (eg. polymerase translocation from
an "A" to another "A" in the template sequence. Furthermore, since
this repeated sequence contains eight Watson-Crick bases instead of
four, it would generate a matrix of 1,312 states instead of
656.
[0196] Some states do not require all variables to be defined (see
above table). For example, characteristics of a nucleotide which
has not yet been incorporated in state 6 do not affect the identity
of state 6.
TABLE-US-00015 577. 6 A X X A
[0197] *The Transition rate between two states will be defined as
such:
TABLE-US-00016 562. 5 T A X C
[0198] P56TAxC=k56TAxC*time_step
Where P56TAxC is the probability of the polymerase completing
translocation from state 5 to state 6 with the additional
nucleotide-template conditions described by "TAxC". K56TAxC is the
transition rate of this translocation.
[0199] Currently in this 656 state system, there are 1568
transition rates to define. There are a number of approximations
that can be made to reduce the number of inputs the user needs to
enter.
[0200] The following combinations may be treated equivalently in
all states transitions: Template nucleotide:
TABLE-US-00017 ACGT TGCA
Likewise, all mismatches may be treated the same K12AT0A=k12xZ0Z
K12CG0T=k12xZ0Z K12CT0T=k12xY0Z K12CT1C=k12xY1Y X=any variable
Y=any mismatch Z=any match
[0201] In this way the user input selection is reduced to
.about.100 unique transition rate variables. All the explicitly
defined rates are automatically assigned the appropriate user
inputs.
[0202] To capitalize on symmetry for the purpose of inserting user
defined transition rates into the matrix automatically, the
organization of the 656-state matrix can be changed:
TABLE-US-00018 Old 1. 1 A A O A 2. 1 A A O C . . . . . . . . . . .
. . . . . . . 656. 7 T T 1 T
TABLE-US-00019 New 1. A 1 A O A 2. A 1 A O C . . . . . . . . . . .
. . . . . . . 656. T 7 T 1 T
[0203] This has two advantages:
[0204] (1.) the template can be extended with only slight
modifications to the matrix. Every Template base in repeated
sequence brings an additional 164 states. Previously, new states
would have to be interwoven into matrix.
[0205] (2) The matrix has a higher degree of symmetry that before,
making it easier to construct the matrix using automated code:
for ii=1:164 eval ([` . . . `]); . . . end % ii Seven "eval"
statements (a function which evaluates an artificially constructed
command) construct seven polymerase states.
[0206] This has been further enhanced to build the matrix for any
given template sequence automatically.
[0207] A further automation of the generation of the state
transition probability matrix is through the building of a
concentration matrix, which contains the concentrations of all
relevant reagents (polymerase, template, nucleotides, etc). This
concentration matrix compliments the rate transition matrix such
that (in the linear concentration limit).
kinetic_matrix=rate_transition_matrix.*conc_matrix
state_transition_probability_matrix=kinetic_matrix*time_step
where each element of the rate transition matrix has been
multiplied by its corresponding dependent variable in the
concentration matrix. In this way we capture the concentration
dependent state transitions (eg. the rate of incorporation of
nucleotides is dependent upon the concentration of nucleotides).
Elements of the matrix which are not concentration dependent are
not changed. Non-linear concentration dependencies may be addressed
using a nonlinear formula defining the kinetic matrix.
[0208] The following describes the state transition probability
matrix:
Matrix=zeros(656,656); Matrix(1, [1,139,577])=[1-p12AA0A-p16AA0A,
p12AA0A, p16AA0A]; Matrix(2, [2,130,578])=[1-p12AA0C-p16AA0C,
p12AA0C, p16AA0C]; . . . Matrix(129,
[129,257,1])=[1-p23AA0A-p21AA0A, p23AA0A, p21AA0A]; . . .
Matrix(656, [656,580,576])=[1-p76TTxT-p75TTxT, p76TTxT, p75TTxT];
where each of the probability values inserted in the matrix have
been calculated using user defined transition rates, concentration
values, and a discrete time step. Note that the first element of a
row is the probability of having no transition between states, and
is thus the difference between 100% and the probabilities of all
state transitions out of that particular state.
[0209] Increasing Efficiency of Simulation:
By raising the state transition probability matrix to a particular
exponential power (eg. 100), we simulate the passage of time of a
particular number of discrete time steps (eg. 100 time steps).
Further improvements to the efficiency of the simulation may be
made through vectorization of many polymerase-template complexes
simultaneously.
1000 { [ template 1 template 2 template 1000 ] 656 * [ 656 .times.
656 kinetic_matrix ] 100 = [ 1000 .times. 656 new state
distributions ] ##EQU00004##
[0210] Speed Limit: DNA synthesis can be tracked by looking at
where pol is on the template.
##STR00006##
[1 0 0 0]="A"
[0 1 0 0]="C"
[0211] etc. . . .
[0212] If we move too fast (i.e. too many time steps in the
transition matrix exponential), the polymerase may go from "A"
straight to "G", making it unclear whether this was forward or
reverse translocation. Therefore an error limit (.about.1e-6) is
set that defines an exponential time factor on the kinetic_matrix.
The speed limit is such that neither the probability of reverse
translocation from "A" to "G" nore the probability of forward
translocation from "A" to "T" exceeds the error rate limit. A
longer DNA repeat sequence will allow us to move faster, but a
repeat sequence which is too long will be computationally
intensive.
[0213] A further application of this program can be the simulation
of reagent consumption rate. Moving at very large step sizes,
polymerase movement is simulated along template. This approach uses
only one template in a continuous distribution of states (instead
of 1000.sup.+ templates in discrete states). This tracks reagent
consumption over time.
[0214] Find the concentration change of reagents based on the
current population of the system and based on the transition rate
constants:
d(dTAP.sub.o).sub.per
pol=C.sub.1.DELTA.tk16AA0A+C.sub.2.DELTA.tk16AA0C+ . . .
C.sub.520.DELTA.tk16TT0T-C.sub.145.DELTA.tk61AA0A-C.sub.146.DELTA.t-
k61AA0C- . . . =C.sub.1p61AA0A+C.sub.146p61AA0C+ . . .
C.sub.145p61AA0A-C.sub.146p61AA0C- . . .
[0215] Where these probabilities are for a 1e.sup.-6 sec time step
from kinetic_matrix: concentration change (Molar) of reagent dTAP
(native) in 1 loop cycle where elapsed time=num_steps*1e.sup.-6
sec
[fast_matrix]=[kinetic_matrix) .sup.num.sup.--.sup.steps Speed
limit:
.DELTA. C max C < 1 % ? ##EQU00005##
Fast matrix=kinetic_matrix.sup.n As N becomes large, the adjustment
to concentrations each loop cycle becomes large and inaccurate.
This is used to set an exponential time factor on the
kinetic_matrix. See FIG. 9, which plots the kinetic matrix jump
size vs. concentration drop.
[0216] Even taking num_steps=1e6 may give accurate "enough"
concentration curves (see the approach to smoothness as step size
decreases).
[0217] The resulting (4096.times.4096 double matrix is a reasonable
memory limit).
[0218] A further application of this program can be the estimation
of the polymerase mismatch fraction using either a continuum model
or counting model. Currently we say that the 2.sup.nd previous
template--nucleotide pair is always a match. (This is to reduce
size of matrix by 4.times. . . . the error should be small unless
there is lots of exonuclease activity).
[0219] Therefore, any forward translocation from state 5 with a
previous mismatch becomes a permanent mismatch (it just won't look
that way if we back up).
forward total translocation
rate=C.sub.5-*C.sub.6--C.sub.6-*k.sub.65 reaction=(mismatch
rate)/(total rate) C.sub.5 represents concentration of all matrix
states with pol in state 5 (see pg. 128) k.sub.56 is the full set
of all corresponding rates for forward translocation forward
mismatch
translocation=C.sub.5.sup.(m).sub.-*k.sub.56.sup.(m)-C.sub.6.sup.(m).sub.-
-*k.sub.65.sup.(m) (In reverse translocation, we never end up in
pol state 5 with previous mismatch, see above).
[0220] We can also make a counting model which counts number of
polymerase/template complexes which have previous
template/nucleotide mismatch and which also do forward
translocation (making mismatch permanent), and average this over
all polymerase to get a mismatch fraction. This should be in the
same ballpark as continuum model estimate above.
1) First set all rate constants equal to T7 polymerase as shown by
Patel, et al. (1991) "Pre-Steady-State Kinetic Analysis of
Processive DNA Replication Including Complete Characterization of
an Exonuclease-Deficient Mutant" Biochemistry 30:511-525.
[0221] Specific rate constants, etc.
K.sub.61.gtoreq.50 .mu.m.sup.-1s.sup.-1 K.sub.12=300
.mu.m.sup.-1s.sup.-1 K.sub.23.gtoreq.9000 .mu.m.sup.-1s.sup.-1
K.sub.34=1200 .mu.m.sup.-1s.sup.-1 K.sub.645>1000
.mu.m.sup.-1s.sup.-1 K.sub.16.gtoreq.1000 .mu.m.sup.-1s.sup.-1
K.sub.21=100 .mu.m.sup.-1s.sup.-1 K.sub.32=18,000
.mu.m.sup.-1s.sup.-1 K.sub.43=18 .mu.m.sup.-1s.sup.-1
K.sub.54.gtoreq.0.54 .mu.m.sup.-1s.sup.-1 (V.sub.max).sub.native=50
bps (V.sub.max).sub.analog=5 bps (k.sub.m).sub.native=0.2 .mu.m
(k.sub.m).sub.analog=6 2) Using dNTP concentration saturation
(.gtoreq.1 mM), set V.sub.max=50 bps by changing k.sub.12
(primarily) and other rate constants (if necessary). Keep all
analog transition rates the same as native dNTP transition rates.
For now cut dissociation (rate.fwdarw.0) 3) Using analog--dNTP
concentration saturation (.gtoreq.1 mM), set V.sub.max=5 by
changing k.sub.45 for analogs only. 4) Set (k.sub.m).sub.native=0.2
.mu.m by setting native dNTP concentration to 0.2 .mu.m and
changing k.sub.61 (natives only) such that V=25 bps. 5) Set
(k.sub.m).sub.native=6 .mu.m by setting analog dNTP concentration
to 6 .mu.m and changing k.sub.m (analogs only) such that V-2.5 bps.
native dNTP's k.sub.61=365 .mu.m.sup.-1s.sup.-1 k.sub.12=60
.mu.m.sup.-1s.sup.-1 k.sub.23=9000 .mu.m.sup.-1s.sup.-1
k.sub.34=1200 .mu.m.sup.-1s.sup.-1 k.sub.45=1000
.mu.m.sup.-1s.sup.-1 k.sub.56=500 .mu.m.sup.-1s.sup.-1 k.sub.16=10
.mu.m.sup.-1s.sup.-1 k.sub.21=100 .mu.m.sup.-1s.sup.-1
k.sub.32=1800 .mu.m.sup.-1s.sup.-1 k.sub.43=18 .mu.m.sup.-1s.sup.-1
k.sub.54=0.5 .mu.m.sup.-1s.sup.-1 k.sub.65=100 .mu.m.sup.-1s.sup.-1
analog dNTP's k.sub.61=1.1 .mu.m.sup.-1s.sup.-1 k.sub.12=60
.mu.m.sup.-1s.sup.-1 k.sub.23=9000 .mu.m.sup.-1s.sup.-1
k.sub.34=5.5 .mu.m.sup.-1s.sup.-1 k.sub.45=5.5 .mu.m.sup.-1s.sup.-1
k.sub.56=500 .mu.m.sup.-1s.sup.-1 k.sub.16=10 .mu.m.sup.-1s.sup.-1
k.sub.21=100 .mu.m.sup.-1s.sup.-1 k.sub.32=800 .mu.m.sup.-1s.sup.-1
k.sub.43=18 .mu.m.sup.-1s.sup.-1 k.sub.54=0.1 .mu.m.sup.-1s.sup.-1
k.sub.65=100 .mu.m.sup.-1s.sup.-1 All rates will be subject to
calibration by future experiments as well. pol_index.m: Initializes
all necessary matrix index lists and pointers based on DNA
sequence. Pol_ratematrix.m: Takes excel file as input, which
contains a list of all unique rate constants, produces transition
rate matrix based on DNA sequence. Pol_conmatrix.m: Takes reagent
concentrations, builds concentration matrix such that: Probability
matrix=time_step*rate_matrix*conc_matrix (for all non-diagonal
elements) Pol_dntp_concumption.m: Calculates reagent consumption
rates based on continuum model. POL_dna.m: Combines all former
functions of POL_DNA, POL_REAGENTS, POL_CURVEMAP, tracks all former
consumption, tracts length distribution of DNA synthesis, tracks
free template, completed dsDNA template, template currently being
worked on, multiple concentration runs possible user defined
repeating DNA sequence, finite length templates pol_metal.m: Full
embodiment of Mg+ depletion experiment, using stripped down version
of POL_DNA.
[0222] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations. All publications, patents, patent applications,
and/or other documents cited in this application are incorporated
by reference in their entirety for all purposes to the same extent
as if each individual publication, patent, patent application,
and/or other document were individually and separately indicated to
be incorporated by reference for all purposes.
Sequence CWU 1
1
641575PRTBacteriophage phi-29 1Met Lys His Met Pro Arg Lys Met Tyr
Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val
Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys
Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val
Gln Ala Asp Leu Tyr Phe His Asn Leu Lys 50 55 60Phe Asp Gly Ala Phe
Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala
Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105
110Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
115 120 125Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly 130 135 140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp
Ile Gln Ile Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln
Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala
Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230
235 240Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala 245 250 255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu 260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu
His Ile Gln His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly
Tyr Ile Pro Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys
Gly Asn Glu Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345
350Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
355 360 365Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Leu 370 375 380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn
Gly Ala Leu Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp
Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr
Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470
475 480Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg 485 490 495Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys 500 505 510Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp
Ile Lys Phe Ser Val 515 520 525Lys Cys Ala Gly Met Thr Asp Lys Ile
Lys Lys Glu Val Thr Phe Glu 530 535 540Asn Phe Lys Val Gly Phe Ser
Arg Lys Met Lys Pro Lys Pro Val Gln545 550 555 560Val Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570
5752862PRTArtificialGST, His, and S-tagged N62D mutant Phi29
polymerase 2Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8603862PRTArtificialGST, His, and S-tagged K135A-N62D mutant Phi29
polymerase 3Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Ala Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8604862PRTArtificialGST, His, and S-tagged E375H-N62D mutant Phi29
polymerase 4Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala
Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp
Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala
Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile
Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly
Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly
His His His His His His Ser Ala Gly Leu Val Pro Arg225 230 235
240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly
Ser Gly 260 265 270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly
Ser Glu Phe Met 275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys
Asp Phe Glu Thr Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp
Ala Tyr Gly Tyr Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr
Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu
Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp
Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360
365Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met
370 375 380Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly
Lys Arg385 390 395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys
Lys Leu Pro Phe Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys
Leu Thr Val Leu Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg
Pro Val Gly Tyr Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile
Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln
Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475
480Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys
485 490 495Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg
Tyr Ala 500 505 510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe
Lys Glu Lys Glu 515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn
Ser Leu Tyr Pro Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr
Gly Glu Pro Ile Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp
Glu Asp Tyr Pro Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe
Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg
Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600
605Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys
610 615 620Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu
Lys Phe625 630 635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile
Asp Lys Trp Thr Tyr 645 650 655Ile Lys Thr Thr Ser His Gly Ala Ile
Lys Gln Leu Ala Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys
Phe Ala Ser Asn Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu
Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu
Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715
720Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr
725 730 735Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr
Gly Thr 740 745 750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro
Lys Lys Leu Gly 755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg
Ala Lys Tyr Leu Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr
Met Lys Glu Val Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro
Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly
Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe
Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840
845Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8605862PRTArtificialGST, His, and S-tagged E375S-N62D mutant Phi29
polymerase 5Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Ser Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8606862PRTArtificialGST, His, and S-tagged E375K-N62D mutant Phi29
polymerase 6Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Lys Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8607862PRTArtificialGST, His, and S-tagged E375R-N62D mutant Phi29
polymerase 7Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Arg Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8608862PRTArtificialGST, His, and S-tagged L384R-N62D mutant Phi29
polymerase 8Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Arg Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val
Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp Tyr Thr
Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
8609862PRTArtificialGST, His, and S-tagged E486A-N62D mutant Phi29
polymerase 9Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710
715 720Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys
Tyr 725 730 735Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu
Thr Gly Thr 740 745 750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp
Pro Lys Lys Leu Gly 755 760 765Tyr Trp Ala His Ala Ser Thr Phe Lys
Arg Ala Lys Tyr Leu Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile
Tyr Met Lys Glu Val Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser
Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala
Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825
830Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val
835 840 845Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys
850 855 86010862PRTArtificialGST, His, and S-tagged E486D-N62D
mutant Phi29 polymerase 10Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile
Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu
Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys
Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu
Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala
Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly
Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val
Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys
Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120
125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala
Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp
Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala
Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile
Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly
Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly
His His His His His His Ser Ala Gly Leu Val Pro Arg225 230 235
240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu
245 250 255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly
Ser Gly 260 265 270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly
Ser Glu Phe Met 275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys
Asp Phe Glu Thr Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp
Ala Tyr Gly Tyr Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr
Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu
Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp
Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360
365Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met
370 375 380Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly
Lys Arg385 390 395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys
Lys Leu Pro Phe Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys
Leu Thr Val Leu Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg
Pro Val Gly Tyr Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile
Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln
Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475
480Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys
485 490 495Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg
Tyr Ala 500 505 510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe
Lys Glu Lys Glu 515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn
Ser Leu Tyr Pro Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr
Gly Glu Pro Ile Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp
Glu Asp Tyr Pro Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe
Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg
Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600
605Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys
610 615 620Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu
Lys Phe625 630 635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile
Asp Lys Trp Thr Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile
Lys Gln Leu Ala Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys
Phe Ala Ser Asn Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu
Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu
Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715
720Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr
725 730 735Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr
Gly Thr 740 745 750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro
Lys Lys Leu Gly 755 760 765Tyr Trp Ala His Asp Ser Thr Phe Lys Arg
Ala Lys Tyr Leu Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr
Met Lys Glu Val Asp Gly Lys Leu785 790 795 800Val Glu Gly Ser Pro
Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly
Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe
Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840
845Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
86011862PRTArtificialGST, His, and S-tagged K512A-N62D mutant Phi29
polymerase 11Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu
Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr
Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn
Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr
Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg
Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys
Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile
Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu
Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu
Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135
140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu
Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala
Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile
Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala
Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp
His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His
His His His His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly
Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250
255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly
260 265 270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu
Phe Met 275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr
Gly Tyr Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile
Gly Asn Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val
Gln Ala Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala
Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser
Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375
380Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys
Arg385 390 395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys
Leu Pro Phe Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu
Thr Val Leu Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro
Val Gly Tyr Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys
Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe
Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser
Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490
495Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala
500 505 510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu
Lys Glu 515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu
Tyr Pro Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu
Pro Ile Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp
Tyr Pro Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu
Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg
Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu
Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615
620Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys
Phe625 630 635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp
Lys Trp Thr Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys
Gln Leu Ala Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe
Ala Ser Asn Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys
Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr
Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr
Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730
735Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr
740 745 750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys
Leu Gly 755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys
Tyr Leu Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys
Glu Val Asp Gly Ala Leu785 790 795 800Val Glu Gly Ser Pro Asp Asp
Tyr Thr Asp Ile Lys Phe Ser Val Lys 805 810 815Cys Ala Gly Met Thr
Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn 820 825 830Phe Lys Val
Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 835 840 845Pro
Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys 850 855
86012845PRTArtificialGST, His, and S-tagged N62D mutant Phi29
polymerase with deletion of residues 505-525 12Met Ser Pro Ile Leu
Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu
Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg
Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu
Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu
Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75
80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr
Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys
Leu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His
Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe
Met Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met Asp
Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys
Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200
205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser
210 215 220Gly Ser Gly His His His His His His Ser Ala Gly Leu Val
Pro Arg225 230 235 240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala
Ala Ala Lys Phe Glu 245 250 255Arg Gln His Met Asp Ser Pro Asp Leu
Gly Thr Gly Gly Gly Ser Gly 260 265 270Asp Asp Asp Asp Lys Ser Pro
Met Gly Tyr Arg Gly Ser Glu Phe Met 275 280 285Lys His Met Pro Arg
Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr Thr 290 295 300Lys Val Glu
Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile Glu305 310 315
320Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Ala
325 330 335Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu
Lys Phe 340 345 350Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn
Gly Phe Lys Trp 355 360 365Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn
Thr Ile Ile Ser Arg Met 370 375 380Gly Gln Trp Tyr Met Ile Asp Ile
Cys Leu Gly Tyr Lys Gly Lys Arg385 390 395 400Lys Ile His Thr Val
Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro 405 410 415Val Lys Lys
Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly Asp 420 425 430Ile
Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro Glu 435 440
445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu
450 455 460Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly
Ser Asp465 470 475 480Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr
Lys Lys Phe Lys Lys 485 490 495Val Phe Pro Thr Leu Ser Leu Gly Leu
Asp Lys Glu Val Arg Tyr Ala 500 505 510Tyr Arg Gly Gly Phe Thr Trp
Leu Asn Asp Arg Phe Lys Glu Lys Glu 515 520 525Ile Gly Glu Gly Met
Val Phe Asp Val Asn Ser Leu Tyr Pro Ala Gln 530 535 540Met Tyr Ser
Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu Gly545 550 555
560Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu
His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu Gly
Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr Lys
Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala Asp
Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu His
Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630 635
640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr Tyr
645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys
Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro
Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala
Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro Val
Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala Arg
Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg Ile
Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745 750Glu
Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly 755 760
765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg Gln
770 775 780Lys Thr Tyr Ile Gln Asp Ile Lys Asp Gly Glu Phe Ser Val
Lys Cys785 790 795 800Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val
Thr Phe Glu Asn Phe 805 810 815Lys Val Gly Phe Ser Arg Lys Met Lys
Pro Lys Pro Val Gln Val Pro 820 825 830Gly Gly Val Val Leu Val Asp
Asp Thr Phe Thr Ile Lys 835 840 84513843PRTArtificialGST, His, and
S-tagged N62D mutant Phi29 polymerase with deletion of residues
505-525 13Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys
Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg
Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr
Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly
Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150
155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys
Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile
Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu
Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly His His His His
His His Ser Ala Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala
Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln
His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265
270Asp Asp Asp Asp Lys Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Met
275 280 285Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr
Thr Thr 290 295 300Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr
Met Asn Ile Glu305 310 315 320Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Ala 325 330 335Trp Val Leu Lys Val Gln Ala
Asp Leu Tyr Phe His Asp Leu Lys Phe 340 345 350Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys Trp 355 360 365Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met 370 375 380Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg385 390
395 400Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe
Pro 405 410 415Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu
Lys Gly Asp 420 425 430Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr
Lys Ile Thr Pro Glu 435 440 445Glu Tyr Ala Tyr Ile Lys Asn Asp Ile
Gln Ile Ile Ala Glu Ala Leu 450 455 460Leu Ile Gln Phe Lys Gln Gly
Leu Asp Arg Met Thr Ala Gly Ser Asp465 470 475 480Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys 485 490 495Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala 500 505
510Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu
515 520 525Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro
Ala Gln 530 535 540Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile
Val Phe Glu Gly545 550 555 560Lys Tyr Val Trp Asp Glu Asp Tyr Pro
Leu His Ile Gln His Ile Arg 565 570 575Cys Glu Phe Glu Leu Lys Glu
Gly Tyr Ile Pro Thr Ile Gln Ile Lys 580 585 590Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly 595 600 605Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met Lys 610 615 620Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe625 630
635 640Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr
Tyr 645 650 655Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala
Lys Leu Met 660 665 670Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn
Pro Asp Val Thr Gly 675 680 685Lys Val Pro Tyr Leu Lys Glu Asn Gly
Ala Leu Gly Phe Arg Leu Gly 690 695 700Glu Glu Glu Thr Lys Asp Pro
Val Tyr Thr Pro Met Gly Val Phe Ile705 710 715 720Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr 725 730 735Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr 740 745
750Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly
755 760 765Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu
Arg Gln 770 775 780Lys Thr Tyr Ile Gln Asp Ile Asp Gly Phe Ser Val
Lys Cys Ala Gly785 790 795 800Met Thr Asp Lys Ile Lys Lys Glu Val
Thr Phe Glu Asn Phe Lys Val 805 810 815Gly Phe Ser Arg Lys Met Lys
Pro Lys Pro Val Gln Val Pro Gly Gly 820 825 830Val Val Leu Val Asp
Asp Thr Phe Thr Ile Lys 835 840147667DNAArtificialplasmide encoding
N62D mutant Phi29 polymerase 14tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg
gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt
ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
tgcaatttat 600tcatatcagg attatcaata ccatattttt gaaaaagccg
tttctgtaat gaaggagaaa 660actcaccgag gcagttccat aggatggcaa
gatcctggta tcggtctgcg attccgactc 720gtccaacatc aatacaacct
attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780aatcaccatg
agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
840agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac 900cgttattcat tcgtgattgc gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac 960aattacaaac aggaatcgaa tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat 1020tttcacctga atcaggatat
tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080tggtgagtaa
ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1140taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
gcaacgctac 1200ctttgccatg tttcagaaac aactctggcg catcgggctt
cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca ttatcgcgag
cccatttata cccatataaa tcagcatcca 1320tgttggaatt taatcgcggc
ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380cccttgtatt
actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680aactctgtag
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1740agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc acacagccca
gcttggagcg aacgacctac 1860accgaactga gatacctaca gcgtgagcta
tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980ccagggggaa
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280tattttctcc
ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2340caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac acccgctgac
gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg tcatcaccga
aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580gtgaagcgat
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt tctgttcatg
ggggtaatga taccgatgaa 2760acgagagagg atgctcacga tacgggttac
tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt aaacaactgg
cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880tcaatgccag
cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2940tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct cgcgtatcgg
tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct agccgggtcc
tcaacgacag gagcacgatc atgctagtca tgccccgcgc 3180ccaccggaag
gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
3240ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt
tccagtcggg 3300aaacctgtcg tgccagctgc attaatgaat cggccaacgc
gcggggagag gcggtttgcg 3360tattgggcgc cagggtggtt tttcttttca
ccagtgagac gggcaacagc tgattgccct 3420tcaccgcctg gccctgagag
agttgcagca agcggtccac gctggtttgc cccagcaggc 3480gaaaatcctg
tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
3540cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta
atggcgcgca 3600ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
agtgggaacg atgccctcat 3660tcagcatttg catggtttgt tgaaaaccgg
acatggcact ccagtcgcct tcccgttccg 3720ctatcggctg aatttgattg
cgagtgagat atttatgcca gccagccaga cgcagacgcg 3780ccgagacaga
acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
3840gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg
ttgatgggtg 3900tctggtcaga gacatcaaga aataacgccg gaacattagt
gcaggcagct tccacagcaa 3960tggcatcctg gtcatccagc ggatagttaa
tgatcagccc actgacgcgt tgcgcgagaa 4020gattgtgcac cgccgcttta
caggcttcga cgccgcttcg ttctaccatc gacaccacca 4080cgctggcacc
cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
4140gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc
gccagttgtt 4200gtgccacgcg gttgggaatg taattcagct ccgccatcgc
cgcttccact ttttcccgcg 4260ttttcgcaga aacgtggctg gcctggttca
ccacgcggga aacggtctga taagagacac 4320cggcatactc tgcgacatcg
tataacgtta ctggtttcac attcaccacc ctgaattgac 4380tctcttccgg
gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
4440ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag
tagtaggttg 4500aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
aggagatggc gcccaacagt 4560cccccggcca cggggcctgc caccataccc
acgccgaaac aagcgctcat gagcccgaag 4620tggcgagccc gatcttcccc
atcggtgatg tcggcgatat aggcgccagc aaccgcacct 4680gtggcgccgg
tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat cgatctcgat
4740cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
ttcccctcta 4800gaaataattt tgtttaactt taagaaggag atatacatat
gtcccctata ctaggttatt 4860ggaaaattaa gggccttgtg caacccactc
gacttctttt ggaatatctt gaagaaaaat 4920atgaagagca tttgtatgag
cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat 4980tgggtttgga
gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt
5040ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
tgtccaaaag 5100agcgtgcaga gatttcaatg cttgaaggag cggttttgga
tattagatac ggtgtttcga 5160gaattgcata tagtaaagac tttgaaactc
tcaaagttga ttttcttagc aagctacctg 5220aaatgctgaa aatgttcgaa
gatcgtttat gtcataaaac atatttaaat ggtgatcatg 5280taacccatcc
tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa
5340tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
gctatcccac 5400aaattgataa gtacttgaaa tccagcaagt atatagcatg
gcctttgcag ggctggcaag 5460ccacgtttgg tggtggcgac catcctccaa
aatcggatgg ttcaactagt ggttctggtc 5520atcaccatca ccatcactcc
gcgggtctgg tgccacgcgg tagtactgca attggtatga 5580aagaaaccgc
tgctgctaaa ttcgaacgcc agcacatgga cagcccagat ctgggtaccg
5640gtggtggctc cggtgatgac gacgacaaga gtcccatggg atatcgggga
tccgaattca 5700tgaagcatat gccgagaaag atgtatagtt gtgactttga
gacaactact aaagtggaag 5760actgtagggt atgggcgtat ggttatatga
atatagaaga tcacagtgag tacaaaatag 5820gtaatagcct ggatgagttt
atggcgtggg tgttgaaggt acaagctgat ctatatttcc 5880atgatctcaa
atttgacgga gcttttatca ttaactggtt ggaacgtaat ggttttaagt
5940ggtcggctga cggattgcca aacacatata atacgatcat atctcgcatg
ggacaatggt 6000acatgattga tatatgttta ggctacaaag ggaaacgtaa
gatacataca gtgatatatg 6060acagcttaaa gaaactaccg tttcctgtta
agaagatagc taaagacttt aaactaactg 6120ttcttaaagg tgatattgat
taccacaaag aaagaccagt cggctataag ataacacccg 6180aagaatacgc
ctatattaaa aacgatattc agattattgc ggaagctctg ttaattcagt
6240ttaagcaagg tttagaccgg atgacagcag gcagtgacag tctaaaaggt
ttcaaggata 6300ttataaccac taagaaattc aaaaaggtgt ttcctacatt
gagtcttgga ctcgataagg 6360aagtgagata cgcctataga ggtggtttta
catggttaaa tgataggttc aaagaaaaag 6420aaatcggaga aggcatggtc
ttcgatgtta atagtctata tcctgcacag atgtatagtc 6480gtctccttcc
atatggtgaa cctatagtat tcgagggtaa atacgtttgg gacgaagatt
6540acccactaca catacagcat atcagatgtg agttcgaatt gaaagagggc
tatataccca 6600ctatacagat aaaaagaagt aggttttata aaggtaatga
gtacctaaaa agtagcggcg 6660gggagatagc cgacctctgg ttgtcaaatg
tagacctaga attaatgaaa gaacactacg 6720atttatataa cgttgaatat
atcagcggct taaaatttaa agcaactaca ggtttgttta 6780aagattttat
agataaatgg acgtacatca agacgacatc agaaggagcg atcaagcaac
6840tagcaaaact gatgttaaac agtctatacg gtaaattcgc tagtaaccct
gatgttacag 6900ggaaagtccc ttatttaaaa gagaatgggg cgctaggttt
cagacttgga gaagaggaaa 6960caaaagaccc tgtttataca cctatgggcg
ttttcatcac tgcatgggct agatacacga 7020caattacagc ggcacaggct
tgttatgatc ggataatata ctgtgatact gacagcatac 7080atttaacggg
tacagagata cctgatgtaa taaaagatat agttgaccct aagaaattgg
7140gatactgggc acatgaaagt acattcaaaa gagctaaata tctgagacag
aagacctata 7200tacaagacat ctatatgaaa gaagtagatg gtaagttagt
agaaggtagt ccagatgatt 7260acactgatat aaaatttagt gttaaatgtg
cgggaatgac tgacaagatt aagaaagagg 7320ttacgtttga gaatttcaaa
gtcggattca gtcggaaaat gaagcctaag cctgtgcaag 7380tgccgggcgg
ggtggttctg gttgatgaca cattcacaat caaataagaa ttctgtacag
7440gccttggcgc gcctgcaggc gagctccgtc gacaagcttg cggccgcact
cgagcaccac 7500caccaccacc accaccacta attgattaat acctaggctg
ctaaacaaag cccgaaagga 7560agctgagttg gctgctgcca ccgctgagca
ataactagca taaccccttg gggcctctaa 7620acgggtcttg aggggttttt
tgctgaaagg aggaactata tccggat 7667151728DNAArtificialencodes
K135A-N62D mutant Phi29 polymerase 15atgaagcaca tgccgagaaa
gatgtatagt tgtgactttg agacaactac taaagtggaa 60gactgtaggg tatgggcgta
tggttatatg aatatagaag atcacagtga gtacaaaata 120ggtaatagcc
tggatgagtt tatggcgtgg gtgttgaagg tacaagctga tctatatttc
180catgatctca aatttgacgg agcttttatc attaactggt tggaacgtaa
tggttttaag 240tggtcggctg acggattgcc aaacacatat aatacgatca
tatctcgcat gggacaatgg
300tacatgattg atatatgttt aggctacaaa gggaaacgta agatacatac
agtgatatat 360gacagcttaa agaaactacc gtttcctgtt aagaagatag
ctgccgactt taaactaact 420gttcttaaag gtgatattga ttaccacaaa
gaaagaccag tcggctataa gataacaccc 480gaagaatacg cctatattaa
aaacgatatt cagattattg cggaagctct gttaattcag 540tttaagcaag
gtttagaccg gatgacagca ggcagtgaca gtctaaaagg tttcaaggat
600attataacca ctaagaaatt caaaaaggtg tttcctacat tgagtcttgg
actcgataag 660gaagtgagat acgcctatag aggtggtttt acatggttaa
atgataggtt caaagaaaaa 720gaaatcggag aaggcatggt cttcgatgtt
aatagtctat atcctgcaca gatgtatagt 780cgtctccttc catacggtga
acctatagta ttcgagggta aatacgtttg ggacgaagat 840tacccactac
acatacagca tatcagatgt gagttcgaat tgaaagaggg ctatataccc
900actatacaga taaaaagaag taggttttat aaaggtaatg agtacctaaa
aagtagcggc 960ggggagatag ccgacctctg gttgtcaaat gtagacctag
aattaatgaa agaacactac 1020gatttatata acgttgaata tatcagcggc
ttaaaattta aagcaactac aggtttgttt 1080aaagatttta tagataaatg
gacgtacatc aagacgacat cagaaggagc gatcaagcaa 1140ctagcaaaac
tgatgttaaa cagtctatac ggtaaattcg ctagtaaccc tgatgttaca
1200gggaaagtcc cttatttaaa agagaatggg gcgctaggtt tcagacttgg
agaagaggaa 1260acaaaagacc ctgtttatac acctatgggc gttttcatca
ctgcatgggc tagatacacg 1320acaattacag cggcacaggc ttgttatgat
cggataatat actgtgatac tgacagcata 1380catttaacgg gtacagagat
acctgatgta ataaaagata tagttgaccc taagaaattg 1440ggatactggg
cacatgaaag tacattcaaa agagctaaat atctgagaca gaagacctat
1500atacaagaca tctatatgaa agaagtagat ggtaagttag tagaaggtag
tccagatgat 1560tacactgata taaaatttag tgttaaatgt gcgggaatga
ctgacaagat taagaaagag 1620gttacgtttg agaatttcaa agtcggattc
agtcggaaaa tgaagcctaa gcctgtgcaa 1680gtgccgggcg gggtggttct
ggttgatgac acattcacaa tcaaataa 1728161728DNAArtificialencodes
E375H-N62D mutant Phi29 polymerase 16atgaagcaca tgccgagaaa
gatgtatagt tgtgactttg agacaactac taaagtggaa 60gactgtaggg tatgggcgta
tggttatatg aatatagaag atcacagtga gtacaaaata 120ggtaatagcc
tggatgagtt tatggcgtgg gtgttgaagg tacaagctga tctatatttc
180catgatctca aatttgacgg agcttttatc attaactggt tggaacgtaa
tggttttaag 240tggtcggctg acggattgcc aaacacatat aatacgatca
tatctcgcat gggacaatgg 300tacatgattg atatatgttt aggctacaaa
gggaaacgta agatacatac agtgatatat 360gacagcttaa agaaactacc
gtttcctgtt aagaagatag ctaaagactt taaactaact 420gttcttaaag
gtgatattga ttaccacaaa gaaagaccag tcggctataa gataacaccc
480gaagaatacg cctatattaa aaacgatatt cagattattg cggaagctct
gttaattcag 540tttaagcaag gtttagaccg gatgacagca ggcagtgaca
gtctaaaagg tttcaaggat 600attataacca ctaagaaatt caaaaaggtg
tttcctacat tgagtcttgg actcgataag 660gaagtgagat acgcctatag
aggtggtttt acatggttaa atgataggtt caaagaaaaa 720gaaatcggag
aaggcatggt cttcgatgtt aatagtctat atcctgcaca gatgtatagt
780cgtctccttc catacggtga acctatagta ttcgagggta aatacgtttg
ggacgaagat 840tacccactac acatacagca tatcagatgt gagttcgaat
tgaaagaggg ctatataccc 900actatacaga taaaaagaag taggttttat
aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag ccgacctctg
gttgtcaaat gtagacctag aattaatgaa agaacactac 1020gatttatata
acgttgaata tatcagcggc ttaaaattta aagcaactac aggtttgttt
1080aaagatttta tagataaatg gacgtacatc aagacgacat cacacggagc
gatcaagcaa 1140ctagcaaaac tgatgttaaa cagtctatac ggtaaattcg
ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa agagaatggg
gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc ctgtttatac
acctatgggc gttttcatca ctgcatgggc tagatacacg 1320acaattacag
cggcacaggc ttgttatgat cggataatat actgtgatac tgacagcata
1380catttaacgg gtacagagat acctgatgta ataaaagata tagttgaccc
taagaaattg 1440ggatactggg cacatgaaag tacattcaaa agagctaaat
atctgagaca gaagacctat 1500atacaagaca tctatatgaa agaagtagat
ggtaagttag tagaaggtag tccagatgat 1560tacactgata taaaatttag
tgttaaatgt gcgggaatga ctgacaagat taagaaagag 1620gttacgtttg
agaatttcaa agtcggattc agtcggaaaa tgaagcctaa gcctgtgcaa
1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728171728DNAArtificialencodes E375S-N62D mutant Phi29 polymerase
17atgaagcaca tgccgagaaa gatgtatagt tgtgactttg agacaactac taaagtggaa
60gactgtaggg tatgggcgta tggttatatg aatatagaag atcacagtga gtacaaaata
120ggtaatagcc tggatgagtt tatggcgtgg gtgttgaagg tacaagctga
tctatatttc 180catgatctca aatttgacgg agcttttatc attaactggt
tggaacgtaa tggttttaag 240tggtcggctg acggattgcc aaacacatat
aatacgatca tatctcgcat gggacaatgg 300tacatgattg atatatgttt
aggctacaaa gggaaacgta agatacatac agtgatatat 360gacagcttaa
agaaactacc gtttcctgtt aagaagatag ctaaagactt taaactaact
420gttcttaaag gtgatattga ttaccacaaa gaaagaccag tcggctataa
gataacaccc 480gaagaatacg cctatattaa aaacgatatt cagattattg
cggaagctct gttaattcag 540tttaagcaag gtttagaccg gatgacagca
ggcagtgaca gtctaaaagg tttcaaggat 600attataacca ctaagaaatt
caaaaaggtg tttcctacat tgagtcttgg actcgataag 660gaagtgagat
acgcctatag aggtggtttt acatggttaa atgataggtt caaagaaaaa
720gaaatcggag aaggcatggt cttcgatgtt aatagtctat atcctgcaca
gatgtatagt 780cgtctccttc catacggtga acctatagta ttcgagggta
aatacgtttg ggacgaagat 840tacccactac acatacagca tatcagatgt
gagttcgaat tgaaagaggg ctatataccc 900actatacaga taaaaagaag
taggttttat aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag
ccgacctctg gttgtcaaat gtagacctag aattaatgaa agaacactac
1020gatttatata acgttgaata tatcagcggc ttaaaattta aagcaactac
aggtttgttt 1080aaagatttta tagataaatg gacgtacatc aagacgacat
caagcggagc gatcaagcaa 1140ctagcaaaac tgatgttaaa cagtctatac
ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa
agagaatggg gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc
ctgtttatac acctatgggc gttttcatca ctgcatgggc tagatacacg
1320acaattacag cggcacaggc ttgttatgat cggataatat actgtgatac
tgacagcata 1380catttaacgg gtacagagat acctgatgta ataaaagata
tagttgaccc taagaaattg 1440ggatactggg cacatgaaag tacattcaaa
agagctaaat atctgagaca gaagacctat 1500atacaagaca tctatatgaa
agaagtagat ggtaagttag tagaaggtag tccagatgat 1560tacactgata
taaaatttag tgttaaatgt gcgggaatga ctgacaagat taagaaagag
1620gttacgtttg agaatttcaa agtcggattc agtcggaaaa tgaagcctaa
gcctgtgcaa 1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728181728DNAArtificialencodes L384R-N62D mutant Phi29 polymerase
18atgaagcaca tgccgagaaa gatgtatagt tgtgactttg agacaactac taaagtggaa
60gactgtaggg tatgggcgta tggttatatg aatatagaag atcacagtga gtacaaaata
120ggtaatagcc tggatgagtt tatggcgtgg gtgttgaagg tacaagctga
tctatatttc 180catgatctca aatttgacgg agcttttatc attaactggt
tggaacgtaa tggttttaag 240tggtcggctg acggattgcc aaacacatat
aatacgatca tatctcgcat gggacaatgg 300tacatgattg atatatgttt
aggctacaaa gggaaacgta agatacatac agtgatatat 360gacagcttaa
agaaactacc gtttcctgtt aagaagatag ctaaagactt taaactaact
420gttcttaaag gtgatattga ttaccacaaa gaaagaccag tcggctataa
gataacaccc 480gaagaatacg cctatattaa aaacgatatt cagattattg
cggaagctct gttaattcag 540tttaagcaag gtttagaccg gatgacagca
ggcagtgaca gtctaaaagg tttcaaggat 600attataacca ctaagaaatt
caaaaaggtg tttcctacat tgagtcttgg actcgataag 660gaagtgagat
acgcctatag aggtggtttt acatggttaa atgataggtt caaagaaaaa
720gaaatcggag aaggcatggt cttcgatgtt aatagtctat atcctgcaca
gatgtatagt 780cgtctccttc catacggtga acctatagta ttcgagggta
aatacgtttg ggacgaagat 840tacccactac acatacagca tatcagatgt
gagttcgaat tgaaagaggg ctatataccc 900actatacaga taaaaagaag
taggttttat aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag
ccgacctctg gttgtcaaat gtagacctag aattaatgaa agaacactac
1020gatttatata acgttgaata tatcagcggc ttaaaattta aagcaactac
aggtttgttt 1080aaagatttta tagataaatg gacgtacatc aagacgacat
cagaaggagc gatcaagcaa 1140ctagcaaaac ggatgttaaa cagtctatac
ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa
agagaatggg gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc
ctgtttatac acctatgggc gttttcatca ctgcatgggc tagatacacg
1320acaattacag cggcacaggc ttgttatgat cggataatat actgtgatac
tgacagcata 1380catttaacgg gtacagagat acctgatgta ataaaagata
tagttgaccc taagaaattg 1440ggatactggg cacatgaaag tacattcaaa
agagctaaat atctgagaca gaagacctat 1500atacaagaca tctatatgaa
agaagtagat ggtaagttag tagaaggtag tccagatgat 1560tacactgata
taaaatttag tgttaaatgt gcgggaatga ctgacaagat taagaaagag
1620gttacgtttg agaatttcaa agtcggattc agtcggaaaa tgaagcctaa
gcctgtgcaa 1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728191728DNAArtificialencodes E486A-N62D mutant Phi29 polymerase
19atgaagcaca tgccgagaaa gatgtatagt tgtgactttg agacaactac taaagtggaa
60gactgtaggg tatgggcgta tggttatatg aatatagaag atcacagtga gtacaaaata
120ggtaatagcc tggatgagtt tatggcgtgg gtgttgaagg tacaagctga
tctatatttc 180catgatctca aatttgacgg agcttttatc attaactggt
tggaacgtaa tggttttaag 240tggtcggctg acggattgcc aaacacatat
aatacgatca tatctcgcat gggacaatgg 300tacatgattg atatatgttt
aggctacaaa gggaaacgta agatacatac agtgatatat 360gacagcttaa
agaaactacc gtttcctgtt aagaagatag ctaaagactt taaactaact
420gttcttaaag gtgatattga ttaccacaaa gaaagaccag tcggctataa
gataacaccc 480gaagaatacg cctatattaa aaacgatatt cagattattg
cggaagctct gttaattcag 540tttaagcaag gtttagaccg gatgacagca
ggcagtgaca gtctaaaagg tttcaaggat 600attataacca ctaagaaatt
caaaaaggtg tttcctacat tgagtcttgg actcgataag 660gaagtgagat
acgcctatag aggtggtttt acatggttaa atgataggtt caaagaaaaa
720gaaatcggag aaggcatggt cttcgatgtt aatagtctat atcctgcaca
gatgtatagt 780cgtctccttc catacggtga acctatagta ttcgagggta
aatacgtttg ggacgaagat 840tacccactac acatacagca tatcagatgt
gagttcgaat tgaaagaggg ctatataccc 900actatacaga taaaaagaag
taggttttat aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag
ccgacctctg gttgtcaaat gtagacctag aattaatgaa agaacactac
1020gatttatata acgttgaata tatcagcggc ttaaaattta aagcaactac
aggtttgttt 1080aaagatttta tagataaatg gacgtacatc aagacgacat
cagaaggagc gatcaagcaa 1140ctagcaaaac tgatgttaaa cagtctatac
ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa
agagaatggg gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc
ctgtttatac acctatgggc gttttcatca ctgcatgggc tagatacacg
1320acaattacag cggcacaggc ttgttatgat cggataatat actgtgatac
tgacagcata 1380catttaacgg gtacagagat acctgatgta ataaaagata
tagttgaccc taagaaattg 1440ggatactggg cacatgccag tacattcaaa
agagctaaat atctgagaca gaagacctat 1500atacaagaca tctatatgaa
agaagtagat ggtaagttag tagaaggtag tccagatgat 1560tacactgata
taaaatttag tgttaaatgt gcgggaatga ctgacaagat taagaaagag
1620gttacgtttg agaatttcaa agtcggattc agtcggaaaa tgaagcctaa
gcctgtgcaa 1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728201728DNAArtificialencodes E486D-N62D mutant Phi29 polymerase
20atgaagcaca tgccgagaaa gatgtatagt tgtgactttg agacaactac taaagtggaa
60gactgtaggg tatgggcgta tggttatatg aatatagaag atcacagtga gtacaaaata
120ggtaatagcc tggatgagtt tatggcgtgg gtgttgaagg tacaagctga
tctatatttc 180catgatctca aatttgacgg agcttttatc attaactggt
tggaacgtaa tggttttaag 240tggtcggctg acggattgcc aaacacatat
aatacgatca tatctcgcat gggacaatgg 300tacatgattg atatatgttt
aggctacaaa gggaaacgta agatacatac agtgatatat 360gacagcttaa
agaaactacc gtttcctgtt aagaagatag ctaaagactt taaactaact
420gttcttaaag gtgatattga ttaccacaaa gaaagaccag tcggctataa
gataacaccc 480gaagaatacg cctatattaa aaacgatatt cagattattg
cggaagctct gttaattcag 540tttaagcaag gtttagaccg gatgacagca
ggcagtgaca gtctaaaagg tttcaaggat 600attataacca ctaagaaatt
caaaaaggtg tttcctacat tgagtcttgg actcgataag 660gaagtgagat
acgcctatag aggtggtttt acatggttaa atgataggtt caaagaaaaa
720gaaatcggag aaggcatggt cttcgatgtt aatagtctat atcctgcaca
gatgtatagt 780cgtctccttc catacggtga acctatagta ttcgagggta
aatacgtttg ggacgaagat 840tacccactac acatacagca tatcagatgt
gagttcgaat tgaaagaggg ctatataccc 900actatacaga taaaaagaag
taggttttat aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag
ccgacctctg gttgtcaaat gtagacctag aattaatgaa agaacactac
1020gatttatata acgttgaata tatcagcggc ttaaaattta aagcaactac
aggtttgttt 1080aaagatttta tagataaatg gacgtacatc aagacgacat
cagaaggagc gatcaagcaa 1140ctagcaaaac tgatgttaaa cagtctatac
ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa
agagaatggg gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc
ctgtttatac acctatgggc gttttcatca ctgcatgggc tagatacacg
1320acaattacag cggcacaggc ttgttatgat cggataatat actgtgatac
tgacagcata 1380catttaacgg gtacagagat acctgatgta ataaaagata
tagttgaccc taagaaattg 1440ggatactggg cacatgacag tacattcaaa
agagctaaat atctgagaca gaagacctat 1500atacaagaca tctatatgaa
agaagtagat ggtaagttag tagaaggtag tccagatgat 1560tacactgata
taaaatttag tgttaaatgt gcgggaatga ctgacaagat taagaaagag
1620gttacgtttg agaatttcaa agtcggattc agtcggaaaa tgaagcctaa
gcctgtgcaa 1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728211728DNAArtificialencodes K512A-N62D mutant Phi29 polymerase
21atgaagcaca tgccgagaaa gatgtatagt tgtgactttg agacaactac taaagtggaa
60gactgtaggg tatgggcgta tggttatatg aatatagaag atcacagtga gtacaaaata
120ggtaatagcc tggatgagtt tatggcgtgg gtgttgaagg tacaagctga
tctatatttc 180catgatctca aatttgacgg agcttttatc attaactggt
tggaacgtaa tggttttaag 240tggtcggctg acggattgcc aaacacatat
aatacgatca tatctcgcat gggacaatgg 300tacatgattg atatatgttt
aggctacaaa gggaaacgta agatacatac agtgatatat 360gacagcttaa
agaaactacc gtttcctgtt aagaagatag ctaaagactt taaactaact
420gttcttaaag gtgatattga ttaccacaaa gaaagaccag tcggctataa
gataacaccc 480gaagaatacg cctatattaa aaacgatatt cagattattg
cggaagctct gttaattcag 540tttaagcaag gtttagaccg gatgacagca
ggcagtgaca gtctaaaagg tttcaaggat 600attataacca ctaagaaatt
caaaaaggtg tttcctacat tgagtcttgg actcgataag 660gaagtgagat
acgcctatag aggtggtttt acatggttaa atgataggtt caaagaaaaa
720gaaatcggag aaggcatggt cttcgatgtt aatagtctat atcctgcaca
gatgtatagt 780cgtctccttc catacggtga acctatagta ttcgagggta
aatacgtttg ggacgaagat 840tacccactac acatacagca tatcagatgt
gagttcgaat tgaaagaggg ctatataccc 900actatacaga taaaaagaag
taggttttat aaaggtaatg agtacctaaa aagtagcggc 960ggggagatag
ccgacctctg gttgtcaaat gtagacctag aattaatgaa agaacactac
1020gatttatata acgttgaata tatcagcggc ttaaaattta aagcaactac
aggtttgttt 1080aaagatttta tagataaatg gacgtacatc aagacgacat
cagaaggagc gatcaagcaa 1140ctagcaaaac tgatgttaaa cagtctatac
ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc cttatttaaa
agagaatggg gcgctaggtt tcagacttgg agaagaggaa 1260acaaaagacc
ctgtttatac acctatgggc gttttcatca ctgcatgggc tagatacacg
1320acaattacag cggcacaggc ttgttatgat cggataatat actgtgatac
tgacagcata 1380catttaacgg gtacagagat acctgatgta ataaaagata
tagttgaccc taagaaattg 1440ggatactggg cacatgaaag tacattcaaa
agagctaaat atctgagaca gaagacctat 1500atacaagaca tctatatgaa
agaagtagat ggtgccttag tagaaggtag tccagatgat 1560tacactgata
taaaatttag tgttaaatgt gcgggaatga ctgacaagat taagaaagag
1620gttacgtttg agaatttcaa agtcggattc agtcggaaaa tgaagcctaa
gcctgtgcaa 1680gtgccgggcg gggtggttct ggttgatgac acattcacaa tcaaataa
1728221677DNAArtificialencodes N62D mutant Phi29 polymerase with
deletion of residues 505-525 22atgaagcaca tgccgagaaa gatgtatagt
tgtgactttg agacaactac taaagtggaa 60gactgtaggg tatgggcgta tggttatatg
aatatagaag atcacagtga gtacaaaata 120ggtaatagcc tggatgagtt
tatggcgtgg gtgttgaagg tacaagctga tctatatttc 180catgatctca
aatttgacgg agcttttatc attaactggt tggaacgtaa tggttttaag
240tggtcggctg acggattgcc aaacacatat aatacgatca tatctcgcat
gggacaatgg 300tacatgattg atatatgttt aggctacaaa gggaaacgta
agatacatac agtgatatat 360gacagcttaa agaaactacc gtttcctgtt
aagaagatag ctaaagactt taaactaact 420gttcttaaag gtgatattga
ttaccacaaa gaaagaccag tcggctataa gataacaccc 480gaagaatacg
cctatattaa aaacgatatt cagattattg cggaagctct gttaattcag
540tttaagcaag gtttagaccg gatgacagca ggcagtgaca gtctaaaagg
tttcaaggat 600attataacca ctaagaaatt caaaaaggtg tttcctacat
tgagtcttgg actcgataag 660gaagtgagat acgcctatag aggtggtttt
acatggttaa atgataggtt caaagaaaaa 720gaaatcggag aaggcatggt
cttcgatgtt aatagtctat atcctgcaca gatgtatagt 780cgtctccttc
catacggtga acctatagta ttcgagggta aatacgtttg ggacgaagat
840tacccactac acatacagca tatcagatgt gagttcgaat tgaaagaggg
ctatataccc 900actatacaga taaaaagaag taggttttat aaaggtaatg
agtacctaaa aagtagcggc 960ggggagatag ccgacctctg gttgtcaaat
gtagacctag aattaatgaa agaacactac 1020gatttatata acgttgaata
tatcagcggc ttaaaattta aagcaactac aggtttgttt 1080aaagatttta
tagataaatg gacgtacatc aagacgacat cagaaggagc gatcaagcaa
1140ctagcaaaac tgatgttaaa cagtctatac ggtaaattcg ctagtaaccc
tgatgttaca 1200gggaaagtcc cttatttaaa agagaatggg gcgctaggtt
tcagacttgg agaagaggaa 1260acaaaagacc ctgtttatac acctatgggc
gttttcatca ctgcatgggc tagatacacg 1320acaattacag cggcacaggc
ttgttatgat cggataatat actgtgatac tgacagcata 1380catttaacgg
gtacagagat acctgatgta ataaaagata tagttgaccc taagaaattg
1440ggatactggg cacatgaaag tacattcaaa agagctaaat atctgagaca
gaagacctat 1500atacaagaca tcaaggatgg agagtttagt gttaaatgtg
cgggaatgac tgacaagatt 1560aagaaagagg ttacgtttga gaatttcaaa
gtcggattca gtcggaaaat gaagcctaag 1620cctgtgcaag tgccgggcgg
ggtggttctg gttgatgaca cattcacaat caaataa
1677231671DNAArtificialencodes N62D mutant Phi29 polymerase with
deletion of residues 505-525 23atgaagcaca tgccgagaaa gatgtatagt
tgtgactttg agacaactac taaagtggaa 60gactgtaggg tatgggcgta tggttatatg
aatatagaag atcacagtga gtacaaaata 120ggtaatagcc tggatgagtt
tatggcgtgg gtgttgaagg tacaagctga tctatatttc 180catgatctca
aatttgacgg agcttttatc attaactggt tggaacgtaa tggttttaag
240tggtcggctg acggattgcc aaacacatat aatacgatca tatctcgcat
gggacaatgg 300tacatgattg atatatgttt aggctacaaa gggaaacgta
agatacatac agtgatatat 360gacagcttaa agaaactacc gtttcctgtt
aagaagatag ctaaagactt taaactaact 420gttcttaaag gtgatattga
ttaccacaaa gaaagaccag tcggctataa gataacaccc 480gaagaatacg
cctatattaa aaacgatatt cagattattg cggaagctct gttaattcag
540tttaagcaag gtttagaccg gatgacagca ggcagtgaca gtctaaaagg
tttcaaggat 600attataacca ctaagaaatt caaaaaggtg tttcctacat
tgagtcttgg actcgataag 660gaagtgagat acgcctatag aggtggtttt
acatggttaa atgataggtt caaagaaaaa 720gaaatcggag aaggcatggt
cttcgatgtt aatagtctat atcctgcaca gatgtatagt 780cgtctccttc
catacggtga acctatagta ttcgagggta aatacgtttg ggacgaagat
840tacccactac acatacagca tatcagatgt gagttcgaat tgaaagaggg
ctatataccc 900actatacaga taaaaagaag taggttttat aaaggtaatg
agtacctaaa aagtagcggc
960ggggagatag ccgacctctg gttgtcaaat gtagacctag aattaatgaa
agaacactac 1020gatttatata acgttgaata tatcagcggc ttaaaattta
aagcaactac aggtttgttt 1080aaagatttta tagataaatg gacgtacatc
aagacgacat cagaaggagc gatcaagcaa 1140ctagcaaaac tgatgttaaa
cagtctatac ggtaaattcg ctagtaaccc tgatgttaca 1200gggaaagtcc
cttatttaaa agagaatggg gcgctaggtt tcagacttgg agaagaggaa
1260acaaaagacc ctgtttatac acctatgggc gttttcatca ctgcatgggc
tagatacacg 1320acaattacag cggcacaggc ttgttatgat cggataatat
actgtgatac tgacagcata 1380catttaacgg gtacagagat acctgatgta
ataaaagata tagttgaccc taagaaattg 1440ggatactggg cacatgaaag
tacattcaaa agagctaaat atctgagaca gaagacctat 1500atacaagaca
tcgacggctt tagtgttaaa tgtgcgggaa tgactgacaa gattaagaaa
1560gaggttacgt ttgagaattt caaagtcgga ttcagtcgga aaatgaagcc
taagcctgtg 1620caagtgccgg gcggggtggt tctggttgat gacacattca
caatcaaata a 167124575PRTArtificialK135A-N62D mutant Phi29
polymerase 24Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly
Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser
Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu
Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp
Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro
Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met
Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His
Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val
Lys Lys Ile Ala Ala Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135
140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr
Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile
Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp
Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys Asp
Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr Leu
Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly
Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230 235 240Glu
Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250
255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu
260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln
His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro
Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu
Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp
Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp
Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala
Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr
Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375
380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val
Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu
Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr
Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr
Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr
Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile Pro
Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470 475 480Gly
Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490
495Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys
500 505 510Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe
Ser Val 515 520 525Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu
Val Thr Phe Glu 530 535 540Asn Phe Lys Val Gly Phe Ser Arg Lys Met
Lys Pro Lys Pro Val Gln545 550 555 560Val Pro Gly Gly Val Val Leu
Val Asp Asp Thr Phe Thr Ile Lys 565 570
57525575PRTArtificialE375H-N62D mutant Phi29 polymerase 25Met Lys
His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr
Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25
30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met
35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu
Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly
Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr
Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu
Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp
Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys
Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His
Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu
Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170
175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser
180 185 190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys
Phe Lys 195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys
Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn
Asp Arg Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val
Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg
Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr
Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg
Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295
300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser
Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp
Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu
Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe
Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser
His Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn Ser
Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly
Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410
415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe
420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln
Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile
His Leu Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile
Val Asp Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His Glu Ser
Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile
Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510Leu Val Glu
Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525Lys
Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535
540Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val
Gln545 550 555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe
Thr Ile Lys 565 570 57526575PRTArtificialE375S-N62D mutant Phi29
polymerase 26Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly
Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser
Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu
Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp
Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro
Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met
Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His
Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val
Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135
140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr
Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile
Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp
Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys Asp
Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr Leu
Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly
Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230 235 240Glu
Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250
255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu
260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln
His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro
Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu
Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp
Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp
Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala
Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr
Ile Lys Thr Thr Ser Ser Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375
380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val
Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu
Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr
Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr
Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr
Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile Pro
Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470 475 480Gly
Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490
495Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys
500 505 510Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe
Ser Val 515 520 525Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu
Val Thr Phe Glu 530 535 540Asn Phe Lys Val Gly Phe Ser Arg Lys Met
Lys Pro Lys Pro Val Gln545 550 555 560Val Pro Gly Gly Val Val Leu
Val Asp Asp Thr Phe Thr Ile Lys 565 570
57527575PRTArtificialE375K-N62D mutant Phi29 polymerase 27Met Lys
His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr
Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25
30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met
35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu
Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly
Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr
Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu
Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp
Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys
Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His
Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu
Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170
175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser
180 185 190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys
Phe Lys 195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys
Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn
Asp Arg Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val
Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg
Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr
Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg
Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295
300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser
Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp
Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu
Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe
Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser
Lys Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn Ser
Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly
Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410
415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe
420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln
Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile
His Leu Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile
Val Asp Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His Glu Ser
Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile
Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510Leu Val Glu
Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525Lys
Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535
540Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val
Gln545 550 555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe
Thr Ile Lys 565 570 57528575PRTArtificialE375R-N62D mutant Phi29
polymerase 28Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp
Ala Tyr Gly Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile
Gly Asn Ser Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln
Ala Asp Leu Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile
Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln
Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg
Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120
125Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly
130 135 140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile
Thr Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln
Ile Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu
Asp Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys
Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr
Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg
Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230 235
240Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala
245 250 255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val
Phe Glu 260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His
Ile Gln His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr
Ile Pro Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly
Asn Glu Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala Asp
Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu His
Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe
Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360
365Tyr Ile Lys Thr Thr Ser Arg Gly Ala Ile Lys Gln Leu Ala Lys Leu
370 375 380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp
Val Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala
Leu Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val
Tyr Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr
Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile
Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile
Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470 475
480Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg
485 490 495Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp
Gly Lys 500 505 510Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile
Lys Phe Ser Val 515 520 525Lys Cys Ala Gly Met Thr Asp Lys Ile Lys
Lys Glu Val Thr Phe Glu 530 535 540Asn Phe Lys Val Gly Phe Ser Arg
Lys Met Lys Pro Lys Pro Val Gln545 550 555 560Val Pro Gly Gly Val
Val Leu Val Asp Asp Thr Phe Thr Ile Lys 565 570
57529575PRTArtificialL384R-N62D mutant Phi29 polymerase 29Met Lys
His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr
Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25
30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met
35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu
Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly
Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr
Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu
Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp
Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys
Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His
Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu
Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170
175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser
180 185 190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys
Phe Lys 195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys
Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn
Asp Arg Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val
Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg
Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr
Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg
Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295
300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser
Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp
Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu
Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe
Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser
Glu Gly Ala Ile Lys Gln Leu Ala Lys Arg 370 375 380Met Leu Asn Ser
Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly
Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410
415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe
420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln
Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile
His Leu Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile
Val Asp Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His Glu Ser
Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile
Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510Leu Val Glu
Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525Lys
Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535
540Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val
Gln545 550 555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe
Thr Ile Lys 565 570 57530575PRTArtificialE486A-N62D mutant Phi29
polymerase 30Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly
Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser
Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu
Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp
Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro
Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met
Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His
Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val
Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135
140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr
Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile
Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp
Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys Asp
Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr Leu
Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly
Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230 235 240Glu
Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250
255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu
260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln
His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro
Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu
Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp
Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp
Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala
Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr
Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375
380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val
Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu
Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr
Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr
Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr
Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile Pro
Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470 475 480Gly
Tyr Trp Ala His Ala Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490
495Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys
500 505 510Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe
Ser Val 515 520 525Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu
Val Thr Phe Glu 530 535 540Asn Phe Lys Val Gly Phe Ser Arg Lys Met
Lys Pro Lys Pro Val Gln545 550 555 560Val Pro Gly Gly Val Val Leu
Val Asp Asp Thr Phe Thr Ile Lys 565 570
57531575PRTArtificialE486D-N62D mutant Phi29 polymerase 31Met Lys
His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr
Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25
30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met
35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu
Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly
Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr
Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu
Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp
Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys
Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His
Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu
Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170
175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser
180 185 190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys
Phe Lys 195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys
Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn
Asp Arg Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val
Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg
Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr
Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg
Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295
300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser
Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp
Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu
Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe
Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser
Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn Ser
Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly
Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410
415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe
420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln
Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile
His Leu Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile
Val Asp Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His Asp Ser
Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile
Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510Leu Val Glu
Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525Lys
Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535
540Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val
Gln545 550 555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe
Thr Ile Lys 565 570 57532575PRTArtificialK512A-N62D mutant Phi29
polymerase 32Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp Phe
Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr Gly
Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn Ser
Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp Leu
Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp
Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro
Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met
Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile His
Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro Val
Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135
140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr
Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile
Ile Ala Glu Ala
165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala
Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr
Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu
Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp
Leu Asn Asp Arg Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly
Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr
Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly
Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280
285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile
290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser
Ser Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val
Asp Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val
Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu
Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr
Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn
Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395
400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu
405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly
Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala
Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp
Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys
Asp Ile Val Asp Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His
Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr
Tyr Ile Gln Asp Ile Tyr Met Lys Glu Val Asp Gly Ala 500 505 510Leu
Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520
525Lys Cys Ala Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu
530 535 540Asn Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro
Val Gln545 550 555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr
Phe Thr Ile Lys 565 570 57533558PRTArtificialN62D mutant Phi29
polymerase with deletion of residues 505-525 33Met Lys His Met Pro
Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr Lys Val Glu
Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30Glu Asp His
Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40 45Ala Trp
Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asp Leu Lys 50 55 60Phe
Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe Lys65 70 75
80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg
85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly
Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu Lys Lys
Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu
Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His Lys Glu Arg Pro
Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile
Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln
Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185 190Asp Ser
Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys 195 200
205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr
210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys
Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn
Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg Leu Leu Pro Tyr
Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr Val Trp Asp Glu
Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg Cys Glu Phe Glu
Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300Lys Arg Ser
Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly305 310 315
320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu Met
325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser Gly
Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe Ile
Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile
Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn Ser Leu Tyr Gly Lys
Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly Lys Val Pro Tyr
Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415Gly Glu Glu
Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425 430Ile
Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys 435 440
445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu Thr Gly
450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp Pro Lys
Lys Leu465 470 475 480Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg
Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile Gln Asp Ile Lys
Asp Gly Glu Phe Ser Val Lys 500 505 510Cys Ala Gly Met Thr Asp Lys
Ile Lys Lys Glu Val Thr Phe Glu Asn 515 520 525Phe Lys Val Gly Phe
Ser Arg Lys Met Lys Pro Lys Pro Val Gln Val 530 535 540Pro Gly Gly
Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys545 550
55534556PRTArtificialN62D mutant Phi29 polymerase with deletion of
residues 505-525 34Met Lys His Met Pro Arg Lys Met Tyr Ser Cys Asp
Phe Glu Thr Thr1 5 10 15Thr Lys Val Glu Asp Cys Arg Val Trp Ala Tyr
Gly Tyr Met Asn Ile 20 25 30Glu Asp His Ser Glu Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met 35 40 45Ala Trp Val Leu Lys Val Gln Ala Asp
Leu Tyr Phe His Asp Leu Lys 50 55 60Phe Asp Gly Ala Phe Ile Ile Asn
Trp Leu Glu Arg Asn Gly Phe Lys65 70 75 80Trp Ser Ala Asp Gly Leu
Pro Asn Thr Tyr Asn Thr Ile Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr
Met Ile Asp Ile Cys Leu Gly Tyr Lys Gly Lys 100 105 110Arg Lys Ile
His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe 115 120 125Pro
Val Lys Lys Ile Ala Lys Asp Phe Lys Leu Thr Val Leu Lys Gly 130 135
140Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly Tyr Lys Ile Thr
Pro145 150 155 160Glu Glu Tyr Ala Tyr Ile Lys Asn Asp Ile Gln Ile
Ile Ala Glu Ala 165 170 175Leu Leu Ile Gln Phe Lys Gln Gly Leu Asp
Arg Met Thr Ala Gly Ser 180 185 190Asp Ser Leu Lys Gly Phe Lys Asp
Ile Ile Thr Thr Lys Lys Phe Lys 195 200 205Lys Val Phe Pro Thr Leu
Ser Leu Gly Leu Asp Lys Glu Val Arg Tyr 210 215 220Ala Tyr Arg Gly
Gly Phe Thr Trp Leu Asn Asp Arg Phe Lys Glu Lys225 230 235 240Glu
Ile Gly Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ala 245 250
255Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu Pro Ile Val Phe Glu
260 265 270Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu His Ile Gln
His Ile 275 280 285Arg Cys Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro
Thr Ile Gln Ile 290 295 300Lys Arg Ser Arg Phe Tyr Lys Gly Asn Glu
Tyr Leu Lys Ser Ser Gly305 310 315 320Gly Glu Ile Ala Asp Leu Trp
Leu Ser Asn Val Asp Leu Glu Leu Met 325 330 335Lys Glu His Tyr Asp
Leu Tyr Asn Val Glu Tyr Ile Ser Gly Leu Lys 340 345 350Phe Lys Ala
Thr Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys Trp Thr 355 360 365Tyr
Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys Gln Leu Ala Lys Leu 370 375
380Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val
Thr385 390 395 400Gly Lys Val Pro Tyr Leu Lys Glu Asn Gly Ala Leu
Gly Phe Arg Leu 405 410 415Gly Glu Glu Glu Thr Lys Asp Pro Val Tyr
Thr Pro Met Gly Val Phe 420 425 430Ile Thr Ala Trp Ala Arg Tyr Thr
Thr Ile Thr Ala Ala Gln Ala Cys 435 440 445Tyr Asp Arg Ile Ile Tyr
Cys Asp Thr Asp Ser Ile His Leu Thr Gly 450 455 460Thr Glu Ile Pro
Asp Val Ile Lys Asp Ile Val Asp Pro Lys Lys Leu465 470 475 480Gly
Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg 485 490
495Gln Lys Thr Tyr Ile Gln Asp Ile Asp Gly Phe Ser Val Lys Cys Ala
500 505 510Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu Asn
Phe Lys 515 520 525Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val
Gln Val Pro Gly 530 535 540Gly Val Val Leu Val Asp Asp Thr Phe Thr
Ile Lys545 550 5553535PRTBacteriophage phi-29 35Thr Tyr Ile Gln Asp
Ile Tyr Met Lys Glu Val Asp Gly Lys Leu Val1 5 10 15Glu Gly Ser Pro
Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys Cys 20 25 30Ala Gly Met
353635PRTBacteriophage B103 36Thr Tyr Ile Gln Asp Ile Tyr Ala Lys
Glu Val Asp Gly Lys Leu Ile1 5 10 15Glu Cys Ser Pro Asp Glu Ala Thr
Thr Thr Lys Phe Ser Val Lys Cys 20 25 30Ala Gly Met
353735PRTBacteriophage PZA 37Thr Tyr Ile Gln Asp Ile Tyr Met Lys
Glu Val Asp Gly Lys Leu Val1 5 10 15Glu Gly Ser Pro Asp Asp Tyr Thr
Thr Ile Lys Phe Ser Val Lys Cys 20 25 30Ala Gly Met
353835PRTBacteriophage M2 38Thr Tyr Ile Gln Asp Ile Tyr Val Lys Glu
Val Asp Gly Lys Leu Lys1 5 10 15Glu Cys Ser Pro Asp Glu Ala Thr Thr
Thr Lys Phe Ser Val Lys Cys 20 25 30Ala Gly Met
353936PRTBacteriophage G1 39Thr Tyr Phe Ile Glu Thr Thr Trp Lys Glu
Asn Asp Lys Gly Lys Leu1 5 10 15Val Val Cys Glu Pro Gln Asp Ala Thr
Lys Val Lys Pro Lys Ile Ala 20 25 30Cys Ala Gly Met
354022PRTBacteriophage CP-1 40Leu Tyr Ile Glu Glu Leu Ile Gln Glu
Asp Gly Thr Thr His Leu Asp1 5 10 15Val Lys Gly Ala Gly Met
204145PRTBacteriophage phi-29 41Leu Lys Phe Lys Ala Thr Thr Gly Leu
Phe Lys Asp Phe Ile Asp Lys1 5 10 15Trp Thr Tyr Ile Lys Thr Thr Ser
Glu Gly Ala Ile Lys Gln Leu Ala 20 25 30Lys Leu Met Leu Asn Ser Leu
Tyr Gly Lys Phe Ala Ser 35 40 454245PRTBacteriophage B103 42Phe Lys
Phe Arg Glu Lys Thr Gly Leu Phe Lys Glu Phe Ile Asp Lys1 5 10 15Trp
Thr Tyr Val Lys Thr His Glu Lys Gly Ala Lys Lys Gln Leu Ala 20 25
30Lys Leu Met Phe Asp Ser Leu Tyr Gly Lys Phe Ala Ser 35 40
454345PRTBacteriophage PZA 43Leu Lys Phe Lys Ala Thr Thr Gly Leu
Phe Lys Asp Phe Ile Asp Lys1 5 10 15Trp Thr His Ile Lys Thr Thr Ser
Glu Gly Ala Ile Lys Gln Leu Ala 20 25 30Lys Leu Met Leu Asn Ser Leu
Tyr Gly Lys Phe Ala Ser 35 40 454445PRTBacteriophage M2 44Phe Lys
Phe Arg Glu Lys Thr Gly Leu Phe Lys Asp Phe Ile Asp Lys1 5 10 15Trp
Thr Tyr Val Lys Thr His Glu Glu Gly Ala Lys Lys Gln Leu Ala 20 25
30Lys Leu Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser 35 40
454547PRTBacteriophage G1 45Phe Met Phe Lys Gly Phe Ile Gly Phe Phe
Asp Glu Tyr Ile Asp Arg1 5 10 15Phe Met Glu Ile Lys Asn Ser Pro Asp
Ser Ser Ala Glu Gln Ser Leu 20 25 30Gln Ala Lys Leu Met Leu Asn Ser
Leu Tyr Gly Lys Phe Ala Thr 35 40 454646PRTBacteriophage CP-1 46Leu
Glu Phe Gln Thr Glu Ser Asp Leu Phe Asp Asp Tyr Ile Thr Thr1 5 10
15Tyr Arg Tyr Lys Lys Glu Asn Ala Gln Ser Pro Ala Glu Lys Gln Lys
20 25 30Ala Lys Ile Met Leu Asn Ser Leu Tyr Gly Lys Phe Gly Ala 35
40 454713PRTBacteriophage phi-29 47Gly Tyr Trp Ala His Glu Ser Thr
Phe Lys Arg Ala Lys1 5 104813PRTBacteriophage B103 48Gly Tyr Trp
Ala His Glu Ser Thr Phe Lys Arg Ala Lys1 5 104913PRTBacteriophage
PZA 49Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg Ala Lys1 5
105013PRTBacteriophage M2 50Gly Tyr Trp Ala His Glu Ser Thr Phe Lys
Arg Ala Lys1 5 105113PRTBacteriophage G1 51Gly Tyr Trp Asp His Glu
Ala Thr Phe Gln Arg Ala Arg1 5 105213PRTBacteriophage CP-1 52Gly
Lys Trp Ala His Glu Gly Arg Ala Val Lys Ala Lys1 5
105310PRTBacteriophage phi-29 53Lys Glu Val Asp Gly Lys Leu Val Glu
Gly1 5 105410PRTBacteriophage B103 54Lys Glu Val Asp Gly Lys Leu
Ile Glu Cys1 5 105510PRTBacteriophage PZA 55Lys Glu Val Asp Gly Lys
Leu Val Glu Gly1 5 105610PRTBacteriophage M2 56Lys Glu Val Asp Gly
Lys Leu Lys Glu Cys1 5 105711PRTBacteriophage G1 57Lys Glu Asn Asp
Lys Gly Lys Leu Val Val Cys1 5 10584PRTBacteriophage CP-1 58Glu Asp
Gly Thr15910PRTBacteriophage phi-29 59Val Lys Lys Ile Ala Lys Asp
Phe Lys Leu1 5 106010PRTBacteriophage B103 60Val Lys Lys Ile Ala
Lys Asp Phe Gln Leu1 5 106110PRTBacteriophage PZA 61Val Lys Lys Ile
Ala Lys Asp Phe Lys Leu1 5 106210PRTBacteriophage M2 62Val Lys Lys
Ile Ala Lys Asp Phe Gln Leu1 5 106310PRTBacteriophage G1 63Val Glu
Gln Ile Ala Lys Gly Phe Gly Leu1 5 106410PRTBacteriophage CP-1
64Ile Ala Thr Met Ala Gly Leu Phe Lys Met1 5 10
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