U.S. patent application number 17/140257 was filed with the patent office on 2021-06-03 for affinity tag nucleic acid and protein compositions and processes for using same.
This patent application is currently assigned to Enzo Biochem, Inc.. The applicant listed for this patent is Enzo Biochem, Inc.. Invention is credited to Juan Carcamo, James J. Donegan, Wayne Patton, Elazar Rabbani, Jannis G. Stavrianopoulos.
Application Number | 20210164017 17/140257 |
Document ID | / |
Family ID | 1000005399899 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210164017 |
Kind Code |
A1 |
Rabbani; Elazar ; et
al. |
June 3, 2021 |
AFFINITY TAG NUCLEIC ACID AND PROTEIN COMPOSITIONS AND PROCESSES
FOR USING SAME
Abstract
The present invention concerns compositions and processes that
use affinity tags for isolating, and detecting or quantifying
analytes, including nucleic acids, proteins and polypeptides.
Compositions include nucleic acid compositions and protein
compositions with affinity binding pairs, including metal binding
peptides and immobilized metals, or peptide affinity groups.
Inventors: |
Rabbani; Elazar; (New York,
NY) ; Stavrianopoulos; Jannis G.; (Bay Shore, NY)
; Donegan; James J.; (Amesbury, MA) ; Patton;
Wayne; (Dix Hills, NY) ; Carcamo; Juan; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Enzo Biochem, Inc. |
New York |
NY |
US |
|
|
Assignee: |
Enzo Biochem, Inc.
New York
NY
|
Family ID: |
1000005399899 |
Appl. No.: |
17/140257 |
Filed: |
January 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16225244 |
Dec 19, 2018 |
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17140257 |
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15209906 |
Jul 14, 2016 |
10196672 |
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16225244 |
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12004842 |
Dec 20, 2007 |
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15209906 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 33/58 20130101; G01N 33/5308 20130101; C12Q 1/6834 20130101;
G01N 2458/10 20130101; C12Q 1/6818 20130101; C12Q 1/6806 20130101;
C12Q 1/6804 20130101 |
International
Class: |
C12Q 1/6804 20180101
C12Q001/6804; G01N 33/58 20060101 G01N033/58; C12Q 1/6806 20180101
C12Q001/6806; C12Q 1/6818 20180101 C12Q001/6818; C12Q 1/6834
20180101 C12Q001/6834; G01N 33/68 20060101 G01N033/68 |
Claims
1. A composition comprising: (i) a fusion antibody comprising an
antibody portion and at least one affinity peptide portion at the
amino terminus or the carboxyl terminus of said antibody portion,
wherein said affinity peptide comprises a metal binding peptide or
is one member of a peptide affinity group; and (ii) a matrix
comprising a metal or a second member of said affinity peptide
group.
2. The composition of claim 1, wherein the at least one affinity
peptide comprises one member of a peptide affinity group and the
matrix comprises the second member of the affinity peptide
group.
3. The composition of claim 2, wherein said peptide affinity group
comprises: S-protein and S-peptide; Glutathione S-Transferase (GST)
tag and Glutathione (GSH); Protein Kinase A catalytic subunit (PKA)
recognition peptide and PKA; Hemagglutinin (HA) epitope tag peptide
and HA; Ketosteroid Isomerase (KSI) tag and oligo Phe; Ketosteroid
Isomerase (KSI) tag and oligo Leu; oligo Arg and oligo Glu; or
oligo Arg and oligo Asp.
4. The composition of claim 1, wherein the affinity peptide
comprises a metal binding peptide and the matrix comprises a
metal.
5. The composition of claim 4, wherein said metal binding peptide
comprises oligohistidine or an oligopeptide comprising the sequence
HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID
NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG
(SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7), KDHLIHNVHKEEHAHAHNK (SEQ
ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO:10), or HGLGHGHEQQHGLGHGH
(SEQ ID NO:9).
6. The composition of claim 1, wherein the fusion antibody is bound
to the matrix via the at least one affinity peptide portion.
7. The composition of claim 2, wherein the fusion antibody is bound
to the matrix via the at least one affinity peptide portion.
8. The composition of claim 3, wherein the fusion antibody is bound
to the matrix via the at least one affinity peptide portion.
9. The composition of claim 4, wherein the fusion antibody is bound
to the matrix via the at least one affinity peptide portion.
10. The composition of claim 5, wherein the fusion antibody is
bound to the matrix via the at least one affinity peptide
portion.
11. A fusion antibody comprising: an antibody portion and at least
one affinity peptide portion at the amino terminus or the carboxyl
terminus of said antibody portion, wherein said affinity peptide
comprises a metal binding peptide or is one member of a peptide
affinity group.
12. The fusion antibody of claim 11, wherein the at least one
affinity peptide comprises one member of a peptide affinity
group.
13. The fusion antibody of claim 12, wherein said peptide affinity
group comprises: S-protein and S-peptide; Glutathione S-Transferase
(GST) tag and Glutathione (GSH); Protein Kinase A catalytic subunit
(PKA) recognition peptide and PKA; Hemagglutinin (HA) epitope tag
peptide and HA; Ketosteroid Isomerase (KSI) tag and oligo Phe;
Ketosteroid Isomerase (KSI) tag and oligo Leu; oligo Arg and oligo
Glu; or oligo Arg and oligo Asp.
14. The fusion antibody of claim 11, wherein the affinity peptide
comprises a metal binding peptide.
15. The fusion antibody of claim 14, wherein said metal binding
peptide comprises oligohistidine or an oligopeptide comprising the
sequence HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG
(SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5),
SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7),
KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID
NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 16/225,244 filed Dec. 19, 2018, which is a divisional of Ser.
No. 15/209,906 filed Jul. 14, 2016, which is a divisional of U.S.
application Ser. No. 12/004,842 filed Dec. 20, 2007, each of which
is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 30, 2020, is named ENZ-79-D1-D1-D1-Application-SL.txt and
is 3,883 bytes in size.
FIELD OF THE INVENTION
[0003] This invention relates to affinity tag compositions
including affinity tag nucleic acids and proteins, and processes
useful for isolating and detecting or quantifying species of a
nucleic acid of interest, and other processes for modifying,
isolating, detecting or quantifying proteins and analytes of
interest.
[0004] All patents, patent applications, patent publications,
scientific articles and the like, cited or identified in this
application are hereby incorporated by reference in their entirety
in order to describe more fully the state of the art to which the
present invention pertains.
BACKGROUND OF THE INVENTION
[0005] For many purposes of manipulating or analyzing nucleic
acids, the first important step is isolation of the nucleic acids
from other cellular material. In this regard, the earliest methods
were relatively crude methods using ethanol precipitation followed
by phase partitioning with organic reagents. For instance, phenol
has been widely used to separate DNA from cellular material while
RNA is more commonly isolated using a guanidinium
isothiocyanate/phenol/chloroform mixture. These methods do not
depend on the particular sequences of the nucleic acids for their
isolation, i.e., they are sequence independent and the basis of
separation is strictly derived from general chemical properties of
DNA and RNA.
[0006] More sophisticated methods were later developed that
employed the particular sequences of the nucleic acids as an
identifying feature for separation, thereby enabling the isolation
of nucleic acids with selected sequences apart from other nucleic
acids as well as from other cellular material. A notable example of
this method is "hybrid capture" where a nucleic acid complementary
to the sequence or sequences of interest is used to specifically
hybridize to one or more target nucleic acids. At a later step, a
tag on the capture probe is used to separate material that has
hybridized to the capture probe from material that remained
unhybridized. Examples of formats that exploit this methodology
include beads with oligo T segments for isolation of polyA RNA, and
strepavidin-coated microtitre plates that can bind biotinylated
primers after amplification reactions. In either case, a moiety
capable of binding the tag is fixed to a solid support, thus
enabling a series of simple washing steps to remove nucleic acids
lacking the sequences of interest. Thus, in one case, a nucleic
acid sequence is added to the capture probe, and in the other case,
one of the nucleotides is modified by the addition of a ligand.
Unfortunately, these methods are disadvantaged by the slower
kinetics of mixed phase hybridization in the first case and the low
capacity engendered by the attachment of large bulky proteins to a
solid matrix in the aforementioned biotin/strepavidin method.
[0007] While conceptually simple, the isolation and purification of
proteins has been at the same time both easier and more
problematic. Unlike nucleic acids that have similar chemical
properties regardless of sequence differences, the variety of
different amino acids and the existence of secondary and tertiary
structures have allowed the application of various criteria to be
used for isolation of a single species of protein. These criteria
include differences in molecular weight, shape, salt solubility,
net charge and polar versus nonpolar characteristics. Thus, for
purification of any given protein, a series of separation steps can
be carried out that will be unique to that particular protein.
However, these standard methods of protein purification lack the
advantages described earlier for isolation of unique nucleic acid
sequences where essentially a single methodology can be applied to
purification of any species of interest. Although this has remained
true for most native proteins, the burgeoning field of recombinant
DNA has allowed more flexibility in modifying desirable proteins
such that they carry additional amino acid sequences that can be
helpful during purification procedures. The most notable example of
such methods is the histidine tag which has been added to either
the carboxy or amino end of the coding sequence (Dobeli et al.,
U.S. Pat. No. 5,284,933). The important feature of this
oligopeptide sequence is that it has an affinity for chelated
metals, such that a matrix with immobilized metal can be used to
bind any protein that has such a histidine tag (Dobeli et al., U.S.
Pat. No. 4,877,830), a method commonly referred to as IMAC
(Immobilized Metal Affinity Chromatography). Thus, a single
isolation procedure can be used for a wide variety of proteins
after the proteins have been suitably modified. Although
oligohistidine is the best known example of an oligo peptide that
can bind to an immobilized metal, other peptides have been
described as well, including one that has the amino acid sequence
HGGHHG (SEQ ID NO:1) (Cheng et al. 2004 Bio-organic & Medicinal
Chemistry Letters 14; 1987-1990).
[0008] The use of non-nucleic acid affinity tags has also been used
in conjunction with nucleic acids. For instance, Min and Verdine
(1996 Nucleic Acids Research 24:3806-3810) have described a nucleic
acid primer with modified bases at the 5' end with histidine
moieties attached to the bases. As such, their primer does not
contain an oligopeptide tag as described above, but rather the 5'
end has been modified with a series of histaminyl purine residues.
Extension of these primers in a PCR reaction allows collection of
the PCR products by means of a chelated resin. No application is
described in this publication, however, for using these constructs
for either signal detection or analyte isolation.
[0009] Stanley et al. (U.S. Pat. No. 5,843,663) describe the use of
affinity agents attached to peptide nucleic acids (PNAs). As
described previously in Min and Verdine (1996), cited supra., the
individual amino acids are attached to each nucleotide analog as
opposed to a true oligohistidine capture agent. It also should be
pointed out that this is not an example of a chimeric molecule
consisting of a nucleic acid and an affinity tag because the
peptide nucleic acid is actually a synthetic substitute for a
nucleic acid. The backbones of the constructs described by Stanley
et al. have an essentially homogeneous nature because both the
subunits of the amino acid segment and the peptide nucleic acid
analogue segment are joined together by a succession of peptide
bonds to form a single polymeric molecule. The method described in
this patent has drawbacks that are intrinsic to the use of peptide
nucleic acids. Specifically, efficient synthesis is limited to only
short PNA sequences and there is a high cost associated with the
reagents used in PNA synthesis.
[0010] Soderlund et al. (U.S. Patent Appl. No. 20040053300)
describe a method of determining the quantity of discrete
polynucleotide analytes by the use of a pool of nucleic acid probes
of various sizes. The probes hybridize to analytes that have been
modified by the addition of an affinity tag (such as oligo
histidine) to the base portion. After hybridization of the probes
to analytes, complexes are isolated by virtue of the presence of
the affinity agent in the analyte allowing binding to a matrix. In
a subsequent step the bound probes are released and quantified,
thus giving an indirect measurement of the amount of analytes
present in a sample. In this particular instance, the analytes
themselves have been covalently attached to an affinity agent.
[0011] Affinity binding pairs have also been used in conjunction
with RNA molecules in Krause and Simmons (U.S. Patent Appl. No.
20060105341). In this application, the use of a so-called RNA
"fusion" molecule with "RNA tags" is described. In this particular
case, however, the "fusion" is not RNA linked to a non-nucleic acid
but rather the molecule is a fusion of different nucleic acid
sequences resulting in a homogenous nucleic acid where a first RNA
segment with a protein binding sequences is appended to a second
RNA segment with a selected nucleic acid sequence. This second RNA
segment may bind, in turn, to a fusion protein with two domains
where one domain binds the RNA tag and the other domain can be an
affinity partner, such as an oligo-His tag, that can be used to
bind the RNA protein complex to a matrix. This composition has been
used for identification and purification of RNA protein complexes
and it has not been used for signal generation or isolation of
nucleic acid analytes.
[0012] Histidine has also been used for other purposes besides an
affinity label. For example, Van Ness et al. (U.S. Pat. No.
7,247,434) describe methods for simultaneously determining a number
of different nucleic acid sequences by the use of tagged nucleic
acid fragments. Sequences are derived from the association of a
different tag for each nucleotide base incorporated into nucleic
acids synthesized from analyte templates. In one particular
instance, a single histidine moiety is used as one of the
base-specific tags where identification is carried out by mass
spectrometry after the nucleic acids have been separated by length.
In this particular instance the histidine is not being used as an
affinity agent but only as an identifier tag.
[0013] Many of the drawback in the previous uses of affinity tags
such as histidine tags are overcome by the present invention.
SUMMARY OF THE INVENTION
[0014] This invention provides a composition which comprises a
nucleic acid and one member of an affinity binding pair, wherein
the member is attached to one or more nucleotides of the nucleic
acid through a phosphate or sugar of the nucleotide or
nucleotides.
[0015] This invention also provides a composition just described
wherein the affinity binding pair comprises: (a) a metal binding
peptide and an immobilized metal, or (b) a peptide affinity
group.
[0016] This invention additionally provides a chimeric nucleic acid
comprising at least two portions, a first portion comprising a
nucleic acid complementary to a nucleic acid sequence of interest,
and a second portion comprising a metal binding peptide, wherein
the metal binding peptide is attached to one or more nucleotides of
the nucleic acid in the first portion through a sugar or phosphate
of the nucleotide or nucleotides.
[0017] Also provided by this invention is a chimeric nucleic acid
comprising at least two portions, a first portion comprising a
nucleic acid complementary to a nucleic acid sequence of interest,
and a second portion comprising one member of a peptide affinity
group, wherein the member is attached to one or more nucleotides of
the nucleic acid in the first portion.
[0018] The present invention provides a process for isolating one
or more species of a nucleic acid of interest. Various steps are
used including the first step of providing a sample containing or
suspected of containing the nucleic acid of interest, a composition
which comprises a nucleic acid portion and a first member of an
affinity binding pair, wherein the nucleic acid portion comprises
sequences complementary to the nucleic acid species of interest,
wherein the affinity binding pair comprises (a) a metal binding
peptide and an immobilized metal, or (b) a peptide affinity group;
and wherein the first member of the affinity binding pair is
attached to one or more nucleotides in the nucleic acid portion;
and a matrix comprising a second member of the affinity binding
pair. The composition hybridizes with any nucleic acid of interest
contained in the sample to form a first complex. The first complex
is contacted with the matrix to form a second complex by means of a
binding interaction between the first member and the second member
of the affinity binding pair. Bound material is separated from
unbound material, thereby isolating the nucleic acid species of
interest.
[0019] The present invention also provides a process for detecting
the presence or quantity of a nucleic acid of interest. In an
initial step, the following elements are provided: a sample
containing labeled nucleic acids, a composition comprising a
nucleic acid portion and a first member of an affinity binding
pair, wherein the nucleic acid portion comprises sequences
complementary to the nucleic acid of interest, and a matrix
comprising a second member of the affinity binding pair; wherein
the affinity binding pair comprises (a) a metal binding peptide and
an immobilized metal, or (b) a peptide affinity group; and wherein
the first member of the affinity binding pair is attached to one or
more nucleotides of the nucleic acid portion. The composition is
allowed to hybridize with any nucleic acid of interest contained in
the sample to form a first complex. This first complex is contacted
with the matrix to form a second complex by means of a binding
interaction between the first member and the second member of the
affinity binding pair. The matrix is washed to remove unhybridized
nucleic acids from the matrix. Detecting or quantifying the nucleic
acid of interest is carried out by means of detecting or
quantifying a signal from the labels.
[0020] The present invention provides yet another process for
detecting the presence or quantity of a nucleic acid of interest.
Various steps are performed including the initial step of providing
the following elements: a sample containing or suspected of
containing the nucleic acid of interest; a labeled probe
complementary to the nucleic acid of interest; a composition
comprising a nucleic acid portion and a first member of an affinity
binding pair, wherein the nucleic acid portion comprises sequences
complementary to the nucleic acid of interest, wherein the affinity
binding pair comprises (a) a metal binding peptide and an
immobilized metal, or (b) a peptide affinity group, and wherein the
first member of the affinity binding pair is attached to one or
more nucleotides of the nucleic acid portion through a sugar,
phosphate or base of the nucleotide or nucleotides; and a matrix
comprising a second member of the affinity binding pair. Any
nucleic acids of interest in the sample are allowed to hybridize
with labeled probe and the composition to form a first complex.
This first complex is contacted with the matrix to form a second
complex by means of a binding interaction between the first member
and the second member of the affinity binding pair. The matrix is
washed to remove unbound materials from the sample. The nucleic
acid of interest is detected or quantified by means of detecting or
quantifying a signal from the labels.
[0021] Yet another process provided by the present invention is one
for detecting the presence or quantity of a nucleic acid of
interest. Various steps are performed including the initial step of
providing a sample containing or suspected of containing nucleic
acid of interest; a probe complementary to the nucleic acid of
interest and comprising two portions, wherein a first comprises
sequences complementary to the nucleic acid of interest, and a
second portion comprising a signal sequence; a composition
comprising a nucleic acid portion and a first member of an affinity
binding pair, wherein the nucleic acid portion comprises sequences
complementary to the nucleic acid of interest, wherein the affinity
binding pair comprises (a) a metal binding peptide and an
immobilized metal, or (b) a peptide affinity group, and wherein the
first member of the affinity binding pair is attached to one or
more nucleotides of the nucleic acid; and a matrix comprising a
second member of the affinity binding pair. Any nucleic acids of
interest in the sample are hybridized with labeled probe and the
composition to form a first complex. The first complex is contacted
with the matrix to form a second complex by means of a binding
interaction between the one or more binding partners and the
affinity peptide. The matrix is washed to remove unbound materials
from the sample. The nucleic acid of interest is detected or
quantified by hybridizing labeled oligonucleotides complementary to
the signal sequence.
[0022] The present invention also provides a fusion protein
comprising a biologically active polypeptide or protein and at
least one affinity peptide attached to the amino-terminus or the
carboxyl-terminus of the biologically active polypeptide or
protein, wherein the affinity peptide comprises at least a portion
of the amino acid sequence of kininogen, such portion comprising a
metal binding peptide.
[0023] The present invention additionally provides a fusion protein
comprising a biologically active polypeptide or protein and an
affinity peptide attached at the amino-terminus and an affinity
peptide attached at the carboxyl-terminus of the biologically
active polypeptide or protein, wherein the affinity peptides
comprise at least a portion of the amino acid sequence kininogen,
such portion comprising a metal binding peptide.
[0024] Another composition provided by this invention is a fusion
protein comprising an antibody linked by its amino- and/or
carboxyl-terminus to one or two affinity peptides, wherein the
affinity peptide binds to a metal, and wherein the antibody has an
affinity to an epitope on a different antibody.
[0025] The invention herein provides a process for modifying a
protein of interest, this process comprising the steps of first
providing (i) a nucleic acid that codes for the protein of
interest; (ii) a nucleic acid that codes for a portion of the amino
acid sequence of kininogen, wherein that portion codes for a metal
binding peptide; and (iii) an expression vector. The nucleic acid
(ii) is added to said nucleic acid (i) to generate a nucleic acid
coding for a fusion protein. The nucleic acid coding for the fusion
protein is inserted into the expression vector (iii), thereby
generating a vector that expresses the modified protein of
interest.
[0026] Additionally the invention herein provides a process for
isolating a protein of interest, and this process comprises an
initial step of providing: (i) a nucleic acid that codes for the
protein of interest; (ii) a nucleic acid that codes for a portion
of the amino acid sequence of kininogen, wherein the portion codes
for a metal binding peptide; (iii) an expression vector; and (iv) a
metal-modified matrix. Other steps include adding the nucleic acid
(ii) to the nucleic acid (i) to generate a nucleic acid coding for
a fusion protein, and inserting the nucleic acid coding for the
fusion protein into the expression vector (iii), thereby generating
a vector that expresses the protein of interest. Finally, the
modified protein of interest is purified by binding the protein of
interest to the metal-modified matrix (iv).
[0027] Other compositions are provided by the present invention
including a fusion protein comprising an antibody and at least one
affinity peptide attached to the amino terminus or the carboxyl
terminus of the antibody, wherein the affinity peptide comprises a
metal binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody.
[0028] Yet another fusion protein provided by this invention is one
comprising an antibody and an affinity peptide attached to the
amino terminus and an affinity peptide attached to carboxyl
terminus of the antibody, wherein the affinity peptide comprises a
metal binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody.
[0029] The invention herein also provides a process for isolating
an analyte of interest, the process comprising the initial step of
providing (i) a sample containing or suspected of containing the
analyte of interest; (ii) a first antibody having an affinity for
the analyte; (iii) a fusion antibody comprising: (a) an antibody
and at least one affinity peptide attached to the amino terminus or
the carboxyl terminus of the antibody, wherein the affinity peptide
comprises a metal binding peptide or is one member of a peptide
affinity group, wherein the antibody has an affinity for a
different antibody; or (b) an antibody and an affinity peptide
attached to the amino terminus and an affinity peptide attached to
carboxyl terminus of the antibody, wherein the affinity peptide
comprises a metal binding peptide or is one member of a peptide
affinity group, wherein the antibody has an affinity for a
different antibody; and (iv) a matrix comprising a metal or a
second member of the affinity peptide group. The sample is
contacted with the first antibody, thereby forming a first complex
between the first antibody and any analyte present in the sample.
The first complex is complexed with the fusion antibody, thereby
forming a second complex between the first complex and the fusion
antibody. The second complex is contacted with the matrix to bind
the second complex to the matrix. Unbound material is removed from
the matrix. The analyte of interest is released from the second
complex, thereby isolating the analyte of interest.
[0030] Another process provided by this invention is one for
detecting or quantifying an analyte of interest, said process
comprising various steps. The first step provides (i) a labeled
sample containing or suspected of containing labeled analyte of
interest; (ii) a first antibody having an affinity for the analyte;
(iii) a fusion antibody comprising: (a) an antibody and at least
one affinity peptide attached to the amino terminus or the carboxyl
terminus of the antibody, wherein the affinity peptide comprises a
metal binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; or
(b) an antibody and an affinity peptide attached to the amino
terminus and an affinity peptide attached to carboxyl terminus of
the antibody, wherein the affinity peptide comprises a metal
binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; and
(iv) a matrix comprising a metal or a second member of the affinity
peptide group. The sample is contacted with the first antibody,
thereby forming a first complex between the first antibody and any
analyte present in the sample. The first complex is contacted with
the fusion antibody, thereby forming a second complex between the
first complex and the fusion antibody. The second complex is
contacted with the matrix to bind the second complex to the matrix.
Unbound material is removed from the matrix. Labeled analytes bound
to the matrix are detected or quantified by means of detecting or
quantifying a signal from the labels.
[0031] Yet another process provided herein is one for detecting or
quantifying an analyte of interest, the process comprising various
steps including the first step of providing (i) a labeled sample
containing or suspected of containing labeled analytes of interest;
(ii) a first antibody having an affinity for the analyte; (iii) a
fusion antibody comprising: (a) an antibody and at least one
affinity peptide attached to the amino terminus or the carboxyl
terminus of the antibody, wherein the affinity peptide comprises a
metal binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; or
(b) an antibody and an affinity peptide attached to the amino
terminus and an affinity peptide attached to carboxyl terminus of
the antibody, wherein the affinity peptide comprises a metal
binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; and
(iv) a matrix comprising a metal or a second member of the affinity
peptide group. A first complex is formed among the matrix, the
fusion antibody and the first antibody. The first complex is
contacted with the labeled sample, thereby forming a second complex
between the first complex and any labeled analytes that may be
present in the sample. Unbound material is removed from the matrix.
The labeled analytes bound to the matrix are detected or quantified
by means of detecting or quantifying a signal from the labels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGS. 1A-F depicts various format that could be used with
chimeric primers.
[0033] FIG. 1A illustrates a chimeric construct with a first
portion consisting of a nucleic acid complementary to a chosen
nucleotide sequence and a second portion with an oligohistidine
portion used to bind nucleic acids with the chosen sequences to a
solid matrix thereby allowing isolation of either nucleic acids
that bind to the matrix or nucleic acids that lack complementarity
to the construct.
[0034] FIG. 1B depicts a format where a chimeric construct similar
to the one in FIG. 1A is used to detect the presence of the
complementary sequence when a collection of labeled analytes are
allowed to hybridize to the construct.
[0035] FIG. 1C shows a chimeric construct similar to the one in
FIG. 1A that is used to detect the presence of unlabeled analytes
by means of a probe complementary to the sequence of interest.
[0036] FIG. 1D illustrates a chimeric construct having energy
transfer elements where hybridization of an analyte labeled with
energy transfer elements provides signal generation that is
dependent upon hybridization of the analyte to the construct.
[0037] FIG. 1E is a depiction of a format where energy transfer
takes place between a labeled analyte and a signal probe.
[0038] FIG. 1F is a depiction of a format where the analyte is
unlabeled and analyte specific energy transfer takes place between
a signal probe and a chimeric construct.
[0039] FIGS. 2A and 2B are illustrative of binding of chimeric
constructs to matrices.
[0040] FIG. 2A shows binding and elution of labelled chimeric
constructs with Ni column.
[0041] FIG. 2B shows binding of labelled chimeric constructs with a
96 well plate.
[0042] FIG. 3 illustrates the effects of various reagents on
binding of chimeric constructs.
DETAILED DESCRIPTION OF THE INVENTION
[0043] This invention provides a composition which comprises a
nucleic acid portion that provides specific hybridization to a
nucleic acid analyte of interest and a non-nucleic acid portion
that comprises at least one member of an affinity binding pair that
allows capture of the composition to a solid matrix wherein the
member is attached to one or more nucleotides of the nucleic acid
and this attachment can be through the phosphate, sugar or base of
the nucleotide or nucleotides. Among such affinity binding pairs
contemplated by this invention are pairs comprising an immobilized
metal and a peptide or oligopeptide that has an affinity for such a
metal.
[0044] Thus, the present invention provides a composition which
comprises a nucleic acid and one member of an affinity binding
pair, wherein the member is attached to one or more nucleotides of
the nucleic acid through a phosphate or sugar of the nucleotide or
nucleotide, and such attachment to such nucleotide or nucleotides
can be through a linker arm as described further below.
Furthermore, in the present composition and invention, the affinity
binding pair comprises: (a) a metal binding peptide and an
immobilized metal, or (b) a peptide affinity group. In a preferred
embodiment the metal is immobilized by chelation. In the present
invention, a peptide or oligopeptide is defined as a succession of
amino acids joined through peptide bonds. Examples of metals that
may be bound by such peptides are nickel, copper, cobalt and zinc.
Examples of such peptides are oligohistidine and an oligopeptide
with the sequence HGGHHG (SEQ ID NO:1) that have been referred to
earlier. Other such oligopeptides that may be of use can include
SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ ID
NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG (SEQ ID NO:6), and
HPHHGGSPHHG (SEQ ID NO:7) described by Pasquinelli et al., 2000
(Biotechnol. Prog. 16, 86-91), KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8)
described by Chaga et al., 1999 (J. Chromatog A. 864; 247-256) as
well as sequences derived from domain 5 of kininogen such as
HGLGHGHEQQHGLGHGH (SEQ ID NO:9) and GHGLGHGHEQQHGLGHGHK [SEQ ID
NO:10] (DeLa Cadena et al., 1992 Protein Science 1; 151-160; Pixley
et al., 2003 J Thrombosis and Haemostasis 1; 1791-1798; and Herwald
et al., 2001 Eur J Biochem 268; 396-404), all of which are
incorporated by reference. Thus, the metal binding peptide can
comprise any of the aforementioned amino acid sequences:
oligohistidine, HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2),
SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID
NO:5), SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7),
KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID
NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
[0045] Other affinity binding pairs that may find use with the
present invention can include peptide affinity pairs. In the
present invention a peptide affinity pair is defined as any binary
combination of peptides, oligopeptides or proteins that that are
capable of recognizing and binding to each other. Examples of such
pairs can include but are not necessarily limited to pairs such as:
S-protein and S-peptide; Glutathione S-Transferase (GST) tag and
Glutathione (GSH); Protein Kinase A catalytic subunit (PKA)
recognition peptide and PKA; Hemagglutinin (HA) epitope tag peptide
and HA, Ketosteroid Isomerase (KSI) tag and oligo Phe; KSI and
oligo Leu; as well as "complementary" pairings such as oligo Arg
with oligo Glu, and oligo Arg with oligo Asp. Thus, as used herein,
the peptide affinity group can comprise: S-protein and S-peptide;
GST and GSH; PKA recognition peptide and PKA; HA peptide epitope
tag and HA; KSI and oligo PHE; KSI and oligo Leu; oligo Arg and
oligo Glu; or oligo Arg and oligo Asp. A more complete discussion
of these binding pairs is included in U.S. Pat. No. 7,183,392,
incorporated herein by reference.
[0046] One member of the affinity binding pair will comprise part
of a chimeric construct joined to a nucleic acid while the
corresponding member of the pair is affixed or immobilized to a
matrix. It is also understood that either member of a pair may be
used as the non-nucleic acid portion such that it can be used with
its corresponding member on the matrix. Thus, for instance, a
chimeric nucleic acid can comprise an oligohistidine portion for
capture by metal chelates attached to a solid matrix (an IMAC
column or plate), or on the other hand, a nucleic acid can be used
that has been modified by the presence of one or more metals
allowing capture on a matrix comprising oligohistidine or some
other metal binding peptide.
[0047] The chimeric nucleic acid provided by the present invention
can comprise at least two portions, a first portion comprising a
nucleic acid complementary to a nucleic acid sequence of interest,
and a second portion comprising a metal binding peptide, e.g.,
nickel, copper, cobalt or zinc. The metal binding peptide can be
attached, desirably through a linker arm as previously described,
to one or more nucleotides of the nucleic acid in the first portion
through a sugar or phosphate of the nucleotide or nucleotides. As
described elsewhere in this disclosure, the metal binding peptide
can comprise any of the amino acid sequences: oligohistidine,
HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID
NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG
(SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7), KDHLIHNVHKEEHAHAHNK (SEQ
ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID NO:10), or HGLGHGHEQQHGLGHGH
(SEQ ID NO:9). One or more energy transfer donors or one or more
energy transfer acceptors can be incorporated into or attached to
the chimeric nucleic acid just described.
[0048] Another chimeric nucleic acid provided by the present
invention comprises at least two portions, a first portion
comprising a nucleic acid complementary to a nucleic acid sequence
of interest, and a second portion comprising one member of a
peptide affinity group. The member can be attached, using a linker
arm desirably, to one or more nucleotides of the nucleic acid in
the first portion. As previously described, the peptide affinity
binding group includes any of the pairs: S-protein and S-peptide,
GST and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo
PHE, KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and
oligo Asp. This additional embodiment of a chimeric nucleic acid
can also further comprise one or more energy transfer donors, or
one or more energy transfer acceptors.
[0049] Synthesis of the chimeric composition can be carried out by
a variety of means where either the base, sugar or phosphate
position of a nucleotide in the nucleic acid portion is used to
attach the affinity agent. Examples of means of modifying nucleic
acids that may be used for this purpose are described in Ward et
al. in U.S. Pat. No. 4,711,955, Engelhardt et al., in U.S. Pat. No.
5,241,060, Stavrianopoulos et al., in U.S. Pat. No. 4,707,440,
Pergolizzi et al., in EP 0 611 828 and Engelhardt et al., in U.S.
Patent Application No. 20030104620, all of which are incorporated
by reference. Attachment can be by means of a covalent attachment
of one of the foregoing metals, oligopeptides or proteins to the
nucleic acid portion, or it may be by means of noncovalent
attachment through a secondary binding pair such as avidin and
biotin. As an example of the latter, one of the proteins described
above as a member of an affinity pair can be biotinylated using
standard methods and the nucleic acid can be covalently linked to
strepavidin. Formation of a complex between these two entities will
create chimeric molecule comprising the affinity member and a
nucleic acid portion.
[0050] Covalent attachment may be direct where the affinity agent
is attached by itself to the nucleic acid portion or it may involve
indirect covalent attachment where there is a linker arm joining
the affinity agent to the nucleic acid portion. The position of
attachment of the non-nucleotide portion to the nucleic acid can
involve any chosen nucleotide; i.e., either internal or terminal
nucleotides are suitable for carrying out the present invention.
Linker arms are well-known in the art and have been described by a
number of authors and researchers. See, for example, Ward et al. in
U.S. Pat. No. 4,711,955, Engelhardt et al., in U.S. Pat. No.
5,241,060, Engelhardt et al., in U.S. Pat. No. 4,894,325, and
Stavrianopoulos et al., in U.S. Pat. No. 7,186,478, all of which
are incorporated herein by reference.
[0051] By means of the present invention, the presence of the
nucleic acid portion will allow the capture of a nucleic acid and
binding of it to a matrix through the affinity agent. The species
of interest can be as broad or as narrow as the user desires by the
appropriate choice of sequences used for the nucleic acid portion.
For instance, the sequence can be selective for a single species
such as a nucleic acid coding for a particular gene, or it may
represent an entire class of molecules. Selectivity can be carried
out with a single sequence in a chimeric composition or there may
be more than one selective sequence in the chimeric composition. It
is also envisioned that selectivity for different sequences may be
carried out either sequentially or in parallel by having different
selective sequences as part of separate chimeric compositions.
[0052] As such, sequences in the nucleic acid portion can comprise
generic sequences such as oligo T or oligo A that can bind to a
wide variety of different nucleic acids or the nucleic acid portion
may comprise unique sequences that will bind to specific mRNA or
cDNA species. An illustration of a possible means of carrying this
out is shown in FIG. 1A. This aspect of the present invention may
be used for either positive or negative selection. As an example of
positive selection, the nucleic acid portion may comprise oligo or
poly T sequences allowing the subsequent binding of polyA mRNA.
Since mRNA generally consists of only 3-6% of total RNA, the
subsequent removal of RNA unable to bind to a matrix bound chimeric
construct results in a powerful enrichment of the poly A sequences
that may be then used for a variety of purposes. As an example of
negative selection, the majority of total RNA consists of rRNA
sequences and these may be removed by the use of chimeric molecules
that comprises sequences complementary to rRNA. After binding of
complexes to a matrix, the portion of the total RNA that contains
mRNA, hnRNA, .mu.RNA and snRNA remains unbound, thereby allowing
any and all of these species to be used in further steps. This may
be of special use and significance when the foregoing analytes are
desirable as labeled nucleic acids and the rRNA itself is of no use
or interest. In such cases, the presence of the rRNA may even be
deleterious since it may consume reagents and contribute noise to
analytic methods, as seen for example, when total RNA is labeled by
photobiotin, 94-97% of the labeled material would be irrelevant to
analysis of polyA mRNA.
[0053] The nucleic acids of the present invention may also be used
in a number of different ways: as part of a detection system; where
a label may be included as part of the composition itself; when the
analyte is being detected or quantified; or when a probe recognizes
the analyte or combinations thereof. For instance, nucleic acid
analytes from biological samples may be labeled directly by
modifying the base, sugar or phosphate moieties. On the other hand,
analytes may also be labeled during the course of copying or in
amplification procedures where labeled nucleotides are provided
during the course of such procedures, thereby synthesizing labeled
complementary or identical copies of the original analytes. A
chimeric composition could be used as a primer to generate a
labeled complementary copy that may be subsequently isolated
afterwards by means of the second member of the affinity pair or a
normal primer could be used preparation of the labeled
complementary copies where a hybridization with a chimeric
composition is carried out afterwards. An example of a copying
reaction that may find use in the present invention could be the
use of samples containing mRNA where labeled cDNA copies are
prepared by means of reverse transcriptase or a DNA polymerase with
reverse transcriptase activity. A general depiction of this type of
format is shown in FIG. 1B.
[0054] In principle, the same methods can be applied to
amplification reactions where there are a series of copying
reactions. Examples of amplification systems that may be useful in
the present invention can include but are not necessarily limited
to the polymerase chain reaction (PCR), ligase chain reaction
(LCR), transcription mediated amplification (TMA), Strand
displacement amplification (SDA), Nucleic acid sequence based
amplification (NASBA) and Secondary Structure Amplification
(Rabbani et al., in U.S. Pat. No. 6,743,605) all of which are
incorporated by reference. Amplifications may be directed towards
specific nucleic acid sequences as is generally used in the
preceding methods, or there may be a more global amplification of
multiple sequences from a library that includes the preceding
methods as well as methods such as those taught by Van Gelder et
al., in U.S. Pat. No. 5,545,522, Kurn in U.S. Pat. No. 6,251,639
and Stavrianopoulos et al., in U.S. Pat. No. 7,163,796, all of
which are incorporated by reference. The synthesis of nucleic acids
may take place after the nucleic acid(s) of interest have been
isolated from a biological sample and released from a matrix or the
reactions may take place while the nucleic acids are still bound to
the matrix. In the latter case, the nucleic acids of the present
invention may be used in a passive manner where they are only used
to immobilize a nucleic acid in an environment where nucleic acid
synthesis reactions may take place. Alternatively, it may be an
active participant where the chimeric nucleic acid comprises a
promoter or acts as a primer in reactions such as those cited
above.
[0055] Detection of an analyte may also take place with unlabeled
analytes by means of the additional use of a labeled probe that is
complementary to the nucleic acid(s) of interest. This may be used
with nucleic acids in their native forms, or complementary copies
derived form copying or amplification procedures. A depiction of a
format with this process is shown in FIG. 1C.
[0056] Other formats are also possible involving energy transfer
elements where either a capture nucleic acid, an analyte or a probe
is labeled with one or more energy transfer donors and one of the
foregoing is labeled with an energy acceptor. Examples of various
formats that could be used with this arrangement are shown in FIGS.
1D-1F. Thus, the compositions of the present invention can comprise
one or more energy transfer donors, or one or more energy transfer
acceptors.
[0057] The present invention and the above-described compositions
can be used to isolate one or more species of a nucleic acid of
interest. In one such process, various elements would be provided
including a sample containing or suspected of containing the
nucleic acid of interest, a composition which comprises a nucleic
acid portion and a first member of an affinity binding pair,
wherein the nucleic acid portion comprises sequences complementary
to the nucleic acid species of interest, and the affinity binding
pair comprises (a) a metal binding peptide and an immobilized
metal, or (b) a peptide affinity group; and wherein the first
member of the affinity binding pair being attached, for example,
through a linker arm, to one or more nucleotides in said nucleic
acid portion; and a matrix comprising a second member of the
affinity binding pair. In this process, the composition hybridizes
with any nucleic acid of interest contained in the sample to form a
first complex. This is followed by contacting the first complex
with the matrix provided to form a second complex by means of a
binding interaction between the first member and the second member
of the affinity binding pair. The material bound to the matrix
could then be separated from unbound material, thereby isolating
said nucleic acid species of interest. Thus, the portion of the
sample that remains bound to the matrix can or may comprise the
nucleic acid species of interest. Contrariwise, the portion of the
sample that remains unbound to the matrix may or could comprise the
nucleic acid species of interest. It should be understood to those
skilled in the art that one or more washing steps could be used in
the process just described above. The metal binding peptide, the
immobilized metal, the peptide affinity group, linker arms, have
been described above with respect to other descriptions of the
present compositions and processes.
[0058] In a different application of the present invention, a
process is provided for detecting the presence or quantity of a
nucleic acid of interest. In this detection or quantification
process, various elements are provided. These include a sample
containing labeled nucleic acids, a composition comprising a
nucleic acid portion and a first member of an affinity binding
pair, wherein the nucleic acid portion comprises sequences
complementary to the nucleic acid of interest, and a solid support
or matrix comprising a second member of the affinity binding pair.
The affinity binding pair can comprise: (a) a metal binding peptide
and an immobilized metal, or (b) a peptide affinity group. The
first member of the affinity binding pair is attached to one or
more nucleotides of said nucleic acid portion, and this attachment
can be through a linker arm as described in further detail above.
Using the elements provided, the above composition is hybridized
with any labeled nucleic acid of interest contained in the sample
to form a first complex. This first complex is contacted with the
matrix to form a second complex by means of a binding interaction
between the first member and the second member of the affinity
binding pair. The matrix is washed one or more times to remove
unhybridized nucleic acids from the matrix. Detection or
quantification of the nucleic acid of interest is carried out by
means of detecting or quantifying a signal from the labels. Such
labels are detectable fluorescently, chemiluminescently,
colorimetrically or enzymatically. The just described process can
include a further step of releasing the second complex from the
matrix prior to detecting or quantifying the nucleic acid of
interest. The nature of the metal binding peptide, the immobilized
metal, the peptide affinity group, the linker arm, energy transfer
donors and energy transfer acceptors have been described earlier in
this disclosure and need not be reiterated here.
[0059] Other processes for detecting or quantifying nucleic acids
of interest are also contemplated and provided by this invention.
In one such detection or quantification process, the following
elements are provided: a sample containing or suspected of
containing the nucleic acid of interest; a labeled probe
complementary to the nucleic acid of interest; a composition
comprising a nucleic acid portion and a first member of an affinity
binding pair, wherein the nucleic acid portion comprises sequences
complementary to the nucleic acid of interest, wherein the affinity
binding pair comprises (a) a metal binding peptide and an
immobilized metal, or (b) a peptide affinity group, and wherein the
first member of the affinity binding pair is attached, e.g.,
through a linker arm, to one or more nucleotides of said nucleic
acid portion through a sugar, phosphate or base of the nucleotide
or nucleotides; and a matrix comprising a second member of the
affinity binding pair. In this process, any nucleic acids of
interest in the sample are hybridized with labeled probe and the
composition to form a first complex. This first complex is
contacted with the matrix to form a second complex by means of a
binding interaction between the first member and the second member
of the affinity binding pair. The matrix can be washed one or more
times to remove unbound materials from the sample. Detection or
quantification of the nucleic acid of interest can be carried out
by means of detecting or quantifying a signal from the labels. Such
labels are detectable fluorescently, chemiluminescently,
colorimetrically or enzymatically. An additional step of releasing
the second complex from the matrix can be carried out or included
in this process prior to carrying out detection or
quantification.
[0060] In the process just described above, aspects such as the
metal binding peptide, the immobilized metal, the peptide affinity
group, the linker arm, energy transfer donors, energy transfer
acceptors, and the like, have been described previously in this
disclosure and will not be reiterated. With respect to the energy
transfer elements, it should be understood that the labeled probes
can comprise one or more energy transfer donors and the composition
can comprise one or more energy transfer acceptors. Alternatively,
the labeled probes can comprise one or more energy transfer
acceptors and the composition can comprise one or more energy
transfer donors. As a different variation, the labeled probes can
comprise one or more energy transfer donors and the nucleic acids
in the sample provided can be labeled with one or more energy
transfer acceptors. Alternatively, in this different variation, the
labeled probes can comprise one or more energy transfer acceptors
and the nucleic acids in the sample provided can be labeled with
one or more energy transfer donors.
[0061] In a different embodiment, the present invention and
compositions can be directed to another process for detecting the
presence or quantity of a nucleic acid of interest. Initially
provided are several elements including: a sample containing or
suspected of containing the nucleic acid of interest; a probe
complementary to the nucleic acid of interest and comprising two
portions, wherein a first comprises sequences complementary to the
nucleic acid of interest, and a second portion comprising a signal
sequence; a composition comprising a nucleic acid portion and a
first member of an affinity binding pair, wherein the nucleic acid
portion comprises sequences complementary to the nucleic acid of
interest, wherein the affinity binding pair comprises (a) a metal
binding peptide and an immobilized metal, or (b) a peptide affinity
group, and wherein the first member of the affinity binding pair is
attached, through a linker arm, for example, to one or more
nucleotides of the nucleic acid; and a matrix comprising a second
member of the affinity binding pair. Any nucleic acids of interest
which are in the sample are allowed to hybridize with the labeled
probe and the composition to form a first complex. Such first
complex is contacted with the matrix to form a second complex by
means of binding interactions between one or more binding partners
and the affinity peptide. The matrix is washed in a single step or
a series of washing steps to remove unbound materials from the
sample. Detection or quantification of the nucleic acid of interest
is carried out by hybridizing labeled oligonucleotides
complementary to the signal sequence. The labeled oligonucleotides
are detectable fluorescently, chemiluminescently, colorimetrically
or enzymatically. The nature of the metal binding peptide, i.e.,
the amino acid sequences used therein, the immobilized metal, the
peptide affinity group, and the like, have been previously
described in this disclosure and will not be repeated here.
[0062] The signal sequence that may be used for this purpose have
been described previously, including methods and compositions
described by Pergolizzi et al., in European Publication No. 0 128
332 A1, based on U.S. patent application Ser. No. 06/491,929, filed
May 5, 1983; and Urdea et al., U.S. Pat. No. 5,124,246. In this
aspect of the present invention, such signal sequence can comprise
a homopolymeric sequence, or it can comprise a heterologous
sequence where the heterologous sequence is neither identical nor
complementary to the nucleic acid of interest.
[0063] In another aspect of the present invention, the use of the
kininogen peptide sequence is disclosed as being useful for
incorporation into nucleic acids coding for proteins of interest.
The provision of this novel affinity peptide may increase the range
of fusion proteins that may be successfully designed with an
affinity sequence. As mentioned earlier, even the flexibility of
being able to use either the carboxy or amino terminus as an
insertion site may be insufficient and both locations may interfere
in either production or activity of the recombinant protein of
interest. The availability of an alternative peptide sequence may
allow generation of recombinant proteins that overcome this
problem.
[0064] Other components or elements can be added to the
just-described composition including one or more energy transfer
donors or one or more energy transfer acceptors.
[0065] As such, this invention is also directed to and provides a
fusion protein comprising a biologically active polypeptide or
protein and at least one affinity peptide attached to the
amino-terminus or the carboxyl-terminus of the biologically active
polypeptide or protein, wherein the affinity peptide comprises at
least a portion of the amino acid sequence of kininogen, the
portion comprising a metal binding peptide. In a different aspect,
the invention also provides a fusion protein comprising a
biologically active polypeptide or protein and an affinity peptide
attached at the amino-terminus and an affinity peptide attached at
the carboxyl-terminus of the biologically active polypeptide or
protein, wherein the affinity peptides comprise at least a portion
of the amino acid sequence kininogen, the portion also comprising a
metal binding peptide. A preferred sequence for the kininogen used
as the affinity peptide in such fusion proteins is
GHGLGHGHEQQHGLGHGHK (SEQ ID NO:10), or a portion thereof. The
kininogen can be human kininogen if desired. Other aspects of the
fusion proteins just described above should be noted. One aspect
relates to the amino acid sequence between the biologically active
polypeptide or protein and the affinity peptides, and this sequence
is or can be recognizable by a protease, such as enterokinase or
coagulation factor Xa. Further, the affinity peptide may bind
nickel, copper, cobalt or zinc.
[0066] A format may be used where the affinity tagged antibody is
specific for a unique target of interest where the target may be a
protein or some other molecule of interest. This approach entails
construction of a unique antibody for each antigen of interest and
it has been previously described in the context of protein arrays
by Wingren et al., (2005 Proteomics 5; 1281-1291) where a library
of single-chain Fv antibodies were fixed to a matrix by either a
metal or an anti-tag antibody. Other antibodies that have been
modified this way have been described by Johnson et al. in U.S.
Patent Application No. 2004/0197866 and Wu et al., in U.S. Patent
Application No. 2006/0094062.
[0067] It should be appreciated that the present invention can be
used to provide a process for modifying proteins of interest. To
modify such a protein, three elements are provided including: (i) a
nucleic acid that codes for the protein of interest; (ii) a nucleic
acid that codes for a portion of the amino acid sequence of
kininogen, wherein the portion codes for a metal binding peptide;
and (iii) an expression vector. To modify the protein with the
elements provided, the nucleic acid (ii) is added to the nucleic
acid (i) to generate a nucleic acid coding for a fusion protein.
The fusion protein coding nucleic acid is inserted into the
expression vector (iii), thereby generating a vector that expresses
the modified protein of interest. Other aspects of the just
described protein modification process deserve mention. One aspect
concerns the expression vector (iii) and it can comprise a number
of different types, including a mammalian expression vector, a
bacterial expression vector, an insect cell expression vector and a
yeast expression vector. The expression vector (iii) can be plasmid
or a viral vector.
[0068] Thus, this invention when applied to the isolation of a
protein of interest, provides the following process. Several
elements are provided including (i) a nucleic acid that codes for
the protein of interest; (ii) a nucleic acid that codes for a
portion of the amino acid sequence of kininogen, wherein that
portion codes for a metal binding peptide; (iii)
[0069] an expression vector; and (iv) a metal-modified matrix. To
isolate the protein of interest, the nucleic acid (ii) is added to
the nucleic acid (i) to generate a nucleic acid coding for a fusion
protein. The nucleic acid coding for the fusion protein is inserted
into the expression vector (iii), resulting in the expression of
the protein of interest. Purification can be carried out by binding
the protein of interest to the metal-modified matrix (iv). This can
be desirably performed using a chromatographic column or a
microtitre plate as the metal-modified matrix. As described
previously, the expression vector (iii) can comprise a mammalian
expression vector, a bacterial expression vector, an insect cell
expression vector or a yeast expression vector. The expression
vector (iii) can also be a plasmid or a viral vector.
[0070] In another embodiment of the present invention, a method of
isolation or detection of proteins is described. As described
earlier, the incorporation of an amino sequence for an affinity tag
has been incorporated into proteins to effect an ease of isolation.
However, this entails a genetic modification of the protein of
interest and it has become clear that even with a flexibility of
being able to add to either the carboxy or the amino end, some
proteins lose functionality by such means. This system does not
allow the detection of unaltered or native proteins. Accordingly,
it is disclosed herein that an antibody to a protein can be
engineered to have an amino sequence that comprises an affinity
peptide, thus allowing capture of the antibody onto a solid matrix
as well as any complex formed between the modified antibody and its
target. This will be of special use and significance when the
target is a protein that is desired to be isolated.
[0071] On the other hand, a more universal reagent can be made by
construction of a tagged antibody that has an affinity for other
antibodies. Thus, for example, an anti-goat antibody that is
derived from mouse cells can be redesigned to comprise an affinity
peptide and used to collect complexes that are made of goat
antibodies that are bound to their particular analyte targets. This
system uses a universal reagent in that only the anti-goat antibody
needs to be modified and this reagent should be able to recognize a
wide variety of complexes formed between goat antibodies and their
antigen targets. Thus, the need for individually modifying each
antibody used for as an antibody/antigen pair is obviated. Although
this method can subsequently be used to isolate the antigen target
by appropriate release of the target, it is understood that the
present invention may also be used in formats that are used to
detect or quantify targets by means of immunoassays. It is
understood that when the terms "antibody" or "antibodies" are used
in the present invention, such terms include, without limitation,
antibody fragments, single chain antibodies, and the like.
[0072] This invention further provides a fusion protein comprising
an antibody and at least one affinity peptide attached to the amino
terminus or the carboxyl terminus of the antibody, wherein the
affinity peptide comprises a metal binding peptide or is one member
of a peptide affinity group. The antibody has an affinity for a
different antibody in this case. A different embodiment provided by
the present invention is a fusion protein comprising an antibody
and an affinity peptide attached to the amino terminus and an
affinity peptide attached to carboxyl terminus of the antibody. The
affinity peptide comprises a metal binding peptide or is one member
of a peptide affinity group, wherein the antibody has an affinity
for a different antibody. In the case of either fusion protein, the
peptide affinity group can comprise S-protein and S-peptide, GST
and GSH, PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE,
KSI and oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo
Asp. Additionally, the metal binding peptide can comprise
oligohistidine or an oligopeptide comprising any of the following
sequences: HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG
(SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID NO:5),
SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7),
KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID
NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
[0073] Processes for analyte isolation and detection or
quantification are also provided by the present invention. To
isolate an analyte of interest, the following process can be used
in accordance with this invention. Four elements are initially
provided, including (i) a sample containing or suspected of
containing the analyte of interest; (ii) a first antibody having an
affinity for the analyte; (iii) a fusion antibody comprising: (a)
an antibody and at least one affinity peptide attached to the amino
terminus or the carboxyl terminus of the antibody, wherein the
affinity peptide comprises a metal binding peptide or is one member
of a peptide affinity group, wherein the antibody has an affinity
for a different antibody; or (b) an antibody and an affinity
peptide attached to the amino terminus and an affinity peptide
attached to carboxyl terminus of the antibody, wherein the affinity
peptide comprises a metal binding peptide or is one member of a
peptide affinity group, wherein the antibody has an affinity for a
different antibody; and (iv) a matrix comprising a metal or a
second member of the affinity peptide group.
[0074] In this analyte isolation process, the sample is allowed to
contact the first antibody, thereby forming a first complex between
the first antibody and any analyte present in the sample. The first
complex is allowed to contact with the fusion antibody, thereby
forming a second complex between the first complex and the fusion
antibody. The second complex is contacted with the matrix to bind
the second complex to the matrix. Unbound material is removed from
the matrix, and the analyte of interest is released from the second
complex, thereby isolating the analyte of interest.
[0075] In this analyte isolation process, other aspects can be
described. For example, the peptide affinity group can comprise
S-protein and S-peptide, GST and GSH, PKA peptide and PKA, HA
peptide and HA, KSI and oligo PHE, KSI and oligo Leu, oligo Arg and
oligo Glu, or oligo Arg and oligo Asp. Further, the metal binding
peptide can comprise oligohistidine or an oligopeptide comprising
any of the sequences: HGGHHG (SEQ ID NO:1), SPHHG (SEQ ID NO:2),
SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ ID NO:4), HPHHGGHPHHG (SEQ ID
NO:5), SPHHGGHPHHG (SEQ ID NO:6), HPHHGGSPHHG (SEQ ID NO:7),
KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8), GHGLGHGHEQQHGLGHGHK (SEQ ID
NO:10), or HGLGHGHEQQHGLGHGH (SEQ ID NO:9).
[0076] Analyte detection or quantification can also be carried out
in accordance with this invention. In a process for detecting or
quantifying an analyte of interest, e.g., a protein or a
polypeptide, the following elements are provided: (i) a labeled
sample containing or suspected of containing labeled analyte of
interest; (ii) a first antibody having an affinity for the analyte;
(iii) a fusion antibody comprising: (a) an antibody and at least
one affinity peptide attached to the amino terminus or the carboxyl
terminus of the antibody, wherein the affinity peptide comprises a
metal binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; or
(b) an antibody and an affinity peptide attached to the amino
terminus and an affinity peptide attached to carboxyl terminus of
the antibody, wherein the affinity peptide comprises a metal
binding peptide or is one member of a peptide affinity group,
wherein the antibody has an affinity for a different antibody; and
(iv) a matrix comprising a metal or a second member of the affinity
peptide group. The sample is contacted with the first antibody,
thereby forming a first complex between the first antibody and any
analyte present in the sample. The first complex so formed is
contacted with the fusion antibody, thereby forming a second
complex between the first complex and the fusion antibody. The
second complex is contacted with the matrix to bind the second
complex to the matrix. Unbound material is removed from the matrix.
Detection or quantification of the labeled analytes bound to the
matrix is performed by means of detecting or quantifying a signal
from the labels. The labels are detectable fluorescently,
chemiluminescently, colorimetrically or enzymatically. It should
also be noted that an additional step can be performed in
connection with this process, namely, the analyte of interest can
be released from the second complex prior to performing any
detection or quantification. As previously described, the peptide
affinity group can comprise S-protein and S-peptide, GST and GSH,
PKA peptide and PKA, HA peptide and HA, KSI and oligo PHE, KSI and
oligo Leu, oligo Arg and oligo Glu, or oligo Arg and oligo Asp.
Further, the metal binding peptide can comprise oligohistidine or
an oligopeptide comprising any of the sequences: HGGHHG (SEQ ID
NO:1), SPHHG (SEQ ID NO:2), SPHHGGSPHHG (SEQ ID NO:3), HPHHG (SEQ
ID NO:4), HPHHGGHPHHG (SEQ ID NO:5), SPHHGGHPHHG (SEQ ID NO:6),
HPHHGGSPHHG (SEQ ID NO:7), KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8),
GHGLGHGHEQQHGLGHGHK (SEQ ID NO:1), or HGLGHGHEQQHGLGHGH (SEQ ID
NO:1).
[0077] This invention also provides a process for detecting and
quantifying an analyte of interest, such as a protein or
polypeptide. In such a process, the following elements are
provided: (i) a labeled sample containing or suspected of
containing labeled analytes of interest; (ii) a first antibody
having an affinity for the analyte; (iii) a fusion antibody
comprising: (a) an antibody and at least one affinity peptide
attached to the amino terminus or the carboxyl terminus of the
antibody, wherein the affinity peptide comprises a metal binding
peptide or is one member of a peptide affinity group, wherein the
antibody has an affinity for a different antibody; or (b) an
antibody and an affinity peptide attached to the amino terminus and
an affinity peptide attached to carboxyl terminus of the antibody,
wherein the affinity peptide comprises a metal binding peptide or
is one member of a peptide affinity group, wherein the antibody has
an affinity for a different antibody; and (iv) a matrix comprising
a metal or a second member of the affinity peptide group. In this
detection or quantification process, a first complex is formed
among the matrix, the fusion antibody and the first antibody. The
first complex is contacted with the labeled sample to form a second
complex between the first complex and any labeled analytes that may
be present in the sample. Unbound material is removed from the
matrix. Labeled analytes bound to the matrix are detected or
quantified by means of detection or quantification of the signal
generated from the labels. Such labels are detectable
fluorescently, chemiluminescently, colorimetrically or
enzymatically. Furthermore, the matrix can comprise an array of
different first antibodies, thereby allowing for detection or
quantification of multiple analytes of interest.
[0078] The examples which follow are set forth to illustrate
various aspects of the present invention but are not intended in
any way to limit its scope as more particularly set forth and
defined in the claims that follow thereafter.
EXAMPLES
[0079] The following are examples illustrating the present
invention.
Example 1 Preparation of Oligonucleotide Modified with
Oligohistidine
Step 1 Synthesis of N-Trifluoroheptahistidine --NHS Ester
[0080] 32 mg (.about.30 .mu.Moles) of acetylated heptahistidine
(Biopeptides, Inc. San Diego Calif.) were dissolved in 200 .mu.l
methanol followed by addition of 400 .mu.l of
methyltrifluoroacetate and 50 .mu.l of pyridine and the mixture
left overnight at room temperature. The liquid phase was evaporated
by a stream of argon and then evaporated in vacuo overnight to
remove any traces of pyridiniumtrifluoroacetate formed by the
presence of trifluoroacetic acid contaminants in the
methyltrifluoroacetate. The residue was dissolved in 200 .mu.l of
dimethylformamide (DMF) followed by the addition of 60 .mu.moles of
n-hydroxysuccinimide and then 50 .mu.l of 0.9 M
dicyclohexylcarbodiimide in DMF. The mixture was stirred overnight
and a urea precipitate was removed by centrifugation.
Step 2 Preparation of Amine Modified Rhodamine Labeled
Oligonucleotide
[0081] A 5' amino modified oligonucleotide was ordered from Sigma
Genosys (Sigma-Aldrich, St. Louis, Mo.) with the following
sequence: 5' amine-tcaaccaac 3'. The oligonucleotide was labeled by
terminal transferase using a 3'-Oligonucleotide labeling system
(Enzo Life Sciences Inc, Farmingdale, N.Y.) and rhodamine labeled
dUTP (Enzo Life Sciences Inc, Farmingdale, N.Y.).
Step 3 Addition of the Oligohistidine Peptide to the Rhodamine
Labeled Oligonucleotide.
[0082] 100 .mu.g of the oligo prepared in step 2 was phenol
extracted, ethanol precipitated and dissolved in 200 .mu.l of 0.2M
Sodium Borate, 5 mM EDTA, pH 8.5 followed by addition of 300 .mu.l
of DMF and 50 .mu.l of the N-trifluoroheptahistidine --NHS ester
synthesized in step 1. The mixture was stirred overnight and the
derivatized DNA was then precipitated by the addition of 10 volumes
of n-butanol. The pellet was dissolved in 200 ul of 1M Lithium
Hydroxide solution and left at room temperature for 30 minutes to
remove the trifluoroacetyl groups. The heptahistidine (SEQ ID
NO:12) modified DNA was then precipitated with 10 volumes of
ethanol and redissolved in binding buffer (20 mM Phosphate, 500 mM
NaCl, pH 7.4)
Example 2 Efficiency of Binding and Release of Oligohisitidine
Modified Oligonucleotide to Nickel Coated Sepharose Beads
(Ni-Column)
[0083] The heptahistidine modified rhodamine oligonucleotide from
Example 1 was diluted in binding buffer (20 mM Phosphate, 500 mM
NaCl, pH 7.4) and added to a Ni-column (Ni Sepharose high
performance, GE Healthcare, Piscataway, N.J.). As a control,
rhodamine labeled oligonucleotide without the addition of the
heptahistidine was also added to a Ni-column. The columns were then
washed with 8 volumes of binding buffer followed by a 5 volumes of
binding buffer containing 0.5 M Imidazole to release the
oligonucleotide histidine groups and the eluants collected.
Quantification was carried out using a spectrofluorometer (Ex: 556
nm, Em: 580) for both the Effluent (Ft) that did not bind and for
the Eluent (Elu) that was released after binding.
[0084] The results of this experiment are shown in FIG. 2(A) as
represented by the percentage of the rhodamine signal of the input
material. In this experiment 75% of the oligohistidine modified
oligonucleotide bound to the column while only 8% of the unmodified
rhodamine oligonucleotide remained bound. The lack of quantitative
binding by the oligohistidine modified preparation is likely to be
an indication that not all of the oligonuclotides were conjugated
to the peptide. This was confirmed by taking the effluent that was
unable to bind and running it a second time over the Ni-column
where the level of binding was observed to be the same as
previously observed with the oligonucleotide lacking the hisitidine
(data not shown).
Example 3 Efficiency of Binding of Oligohistidine Modified
Oligonucleotide to Nickel Coated 96-Well Plates
[0085] Binding of the oligohistidine modified oligonucleotide to a
matrix was also tested by binding to 96 well plates instead of the
columns used in Example 2. Identical dilutions of histidine
modified and unmodified rhodamine labeled oligonucleotides from
Example 2 were added to Nickel-coated plates (HisGrab Nickel coated
96-well plates, Pierce, Rockford, Ill.) and incubated for 3 hours
at room temperature, followed by washing 3 times with 200 ul
binding buffer. The bound DNA was measured by detecting rhodamine
with a plate reader (filters: Ex550, Em610, Fluostar Optima, BMG
Labtech). Results of this experiment are shown in FIG. 2 (B). The
Histidine-modified DNA bound to the Ni-plates more effectively than
the control DNA. For example, an input of 10 ul of control and
His(7)-DNA showed 90% of the histidine modified DNA being bound to
the wells, while only 10% of the control DNA was detected. The
results shown in FIG. 2 (B) also indicate that with the highest
input level (50 ul), the wells were overloaded.
Example 4 Effects of Other Reagents on Binding
[0086] Preparations of nucleic acids are commonly taken up in the
presence of chelators such as EDTA or SSC (standard saline
citrate). Since it is possible that these could be competitors for
a peptide/chelate interaction, the oligonucleotides from Example 1
were tested for the ability to be bound in their presence. The
histidine modified oligos were incubated with Ni beads in the
presence of binding buffer (control), or binding buffer with either
0.5 mM EDTA or 1.times.SSC. The results of this Experiment are
shown in FIG. 3, where it can be seen that the presence of at least
low levels of these components had no effect on the efficiency of
binding of the oligohistidine modified nucleic acid.
[0087] Many obvious variations will be suggested to those of
ordinary skill in the art in light of the above detailed
descriptions of the present invention. All such obvious variations
are fully contemplated and are embraced by the scope and spirit of
the present invention as set forth in the claims that now follow.
Sequence CWU 1
1
1216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1His Gly Gly His His Gly1 525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Ser
Pro His His Gly1 5311PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Ser Pro His His Gly Gly Ser
Pro His His Gly1 5 1045PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 4His Pro His His Gly1
5511PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5His Pro His His Gly Gly His Pro His His Gly1 5
10611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ser Pro His His Gly Gly His Pro His His Gly1 5
10711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7His Pro His His Gly Gly Ser Pro His His Gly1 5
10819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Lys Asp His Leu Ile His Asn Val His Lys Glu Glu
His Ala His Ala1 5 10 15His Asn Lys917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9His
Gly Leu Gly His Gly His Glu Gln Gln His Gly Leu Gly His Gly1 5 10
15His1019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gly His Gly Leu Gly His Gly His Glu Gln Gln His
Gly Leu Gly His1 5 10 15Gly His Lys116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11His
His His His His His1 5127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12His His His His His His
His1 5
* * * * *