U.S. patent application number 12/842764 was filed with the patent office on 2012-06-21 for affinity peptides and method for purification of recombinant proteins.
This patent application is currently assigned to SIGMA-ALDRICH CO.. Invention is credited to Ian R. Brockie, Ronald A. Hernan, Elizabeth Jenkins, Richard J. Mehigh.
Application Number | 20120157659 12/842764 |
Document ID | / |
Family ID | 31498502 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120157659 |
Kind Code |
A1 |
Hernan; Ronald A. ; et
al. |
June 21, 2012 |
AFFINITY PEPTIDES AND METHOD FOR PURIFICATION OF RECOMBINANT
PROTEINS
Abstract
This invention describes a process for separating a fusion
protein or polypeptide in the form of its precursor from a mixture
containing said fusion protein and impurities, which comprises
contacting said fusion protein with a resin containing immobilized
metal ions, said fusion protein covalently operably linked directly
or indirectly to an immobilized metal ion-affinity peptide, binding
said fusion protein to said resin, and selectively eluting said
fusion protein from said resin.
Inventors: |
Hernan; Ronald A.; (Ballwin,
MO) ; Mehigh; Richard J.; (St. Louis, MO) ;
Brockie; Ian R.; (St. Louis, MO) ; Jenkins;
Elizabeth; (Sherman, IL) |
Assignee: |
SIGMA-ALDRICH CO.
St. Louis
MO
|
Family ID: |
31498502 |
Appl. No.: |
12/842764 |
Filed: |
July 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10460524 |
Jun 12, 2003 |
7799561 |
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12842764 |
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60388059 |
Jun 12, 2002 |
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Current U.S.
Class: |
530/324 ;
530/325; 530/326; 530/327; 530/328; 530/350 |
Current CPC
Class: |
B01J 20/3265 20130101;
C07K 1/047 20130101; C07K 1/22 20130101; C07K 7/06 20130101; B01J
20/286 20130101; B01D 15/3828 20130101; C07K 7/08 20130101 |
Class at
Publication: |
530/324 ;
530/325; 530/326; 530/327; 530/328; 530/350 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 14/00 20060101 C07K014/00; C07K 7/08 20060101
C07K007/08 |
Claims
1. A polypeptide, protein or protein fragment represented by the
formula
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-His)-Sp.sub.2-R.sub.2,
wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide, R.sub.1 is hydrogen, a polypeptide,
protein or protein fragment, Sp.sub.1 is a covalent bond or a
spacer comprising at least one amino acid residue, R.sub.2 is
hydrogen, a polypeptide, protein or protein fragment, Sp.sub.2 is a
covalent bond or a spacer comprising at least one amino acid
residue, Z.sub.1 is an amino acid residue selected from the group
consisting of Ala, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro, Ser,
Thr, Trp, and Val, and Z.sub.2 is an amino acid residue selected
from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly,
Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val.
2. The polypeptide, protein or protein fragment of claim 1, wherein
Z.sub.1 is selected from the group consisting of Ala, Asn, Ile,
Lys, Phe, Ser, Thr, and Val, and Z.sub.2 is selected from the group
consisting of Ala, Asn, Gly, Lys, Ser, Thr and Tyr.
3. The polypeptide, protein or protein fragment of claim 1, wherein
Z.sub.1 and Z.sub.2 are selected from the group consisting of: (a)
Z.sub.1 is Asn and Z.sub.2 is Gly; (b) Z.sub.1 is Asn and Z.sub.2
is Lys (c) Z.sub.1 is Lys and Z.sub.2 is Gly. (d) Z.sub.1 is Lys
and Z.sub.2 is Lys. (e) Z.sub.1 is Ile and Z.sub.2 is Asn; (f)
Z.sub.1 is Thr and Z.sub.2 is Ser; (g) Z.sub.1 is Ser and Z.sub.2
is Tyr; (h) Z.sub.1 is Val and Z.sub.2 is Ala; and (i) Z.sub.1 is
Ala and Z.sub.2 is Lys.
4. The polypeptide, protein or protein fragment of claim 1, wherein
R.sub.1 or R.sub.2 is hydrogen.
5. The polypeptide, protein or protein fragment of claim 1, wherein
R.sub.1 or R.sub.2 is an amino acid residue.
6. The polypeptide, protein or protein fragment of claim 1, wherein
Sp.sub.1 or Sp.sub.2 is a spacer comprising a proteolytic cleavage
site, a fusion protein, a secretion sequence, a leader sequence for
cellular targeting an antibody epitope or an internal ribosomal
sequences.
7. The polypeptide, protein or protein fragment of claim 1, wherein
Sp.sub.1 or Sp.sub.2 is a spacer comprising a proteolytic cleavage
site.
8. The polypeptide, protein or protein fragment of claim 7, wherein
the proteolytic cleavage site is cleaved with enterokinase.
9. The polypeptide, protein or protein fragment of claim 1, wherein
any one of Sp.sub.1, Sp.sub.2, R.sub.1 and R.sub.2 comprises at
least one of the amino acid sequences selected from the group
consisting of SEQ ID NOS: 1-17.
10. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sub.1 or Sp.sub.2 is a spacer comprising the enzyme
glutathione-S-transferase of the parasite helminth Schistosoma
japonicum.
11. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sub.1 or Sp.sub.2 is a spacer comprising the amino acid
sequence DYKDDDDK (SEQ ID NO: 15).
12. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sub.1 or Sp.sub.2 is a spacer comprising the amino acid
sequence DLYDDDDK (SEQ ID NO: 16).
13. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sub.1 or Sp.sub.2 is a spacer comprising the amino acid
sequence
Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-A-
sp-Asp-Asp-Asp-Lys (SEQ ID NO: 17).
14. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sup.1 or Sp.sup.2 is a spacer comprising at least one
amino acid residue, said spacer comprising an antigenic domain,
wherein the antigenic domain comprises the sequence TABLE-US-00001
(SEQ ID NO: 39)
X.sup.20-(X.sup.1-Y-K-X.sup.2-X.sup.3-D-X.sup.4).sub.n-X.sup.5-(X.sup.1-Y--
K-X.sup.7-X.sup.8-D-X.sup.9-K)-X.sup.21
where: D, Y and K are their representative amino acids; X.sup.20
and X.sup.21 are independently a hydrogen or a covalent bond; each
X.sup.1 and X.sup.4 is independently a covalent bond or at least
one amino acid residue selected from the group consisting of
aromatic amino acid residues and hydrophilic amino acid residues;
each X.sup.2, X.sup.3, X.sup.7 and X.sup.8 is independently an
amino acid residue selected from the group consisting of aromatic
amino acid residues and hydrophilic amino acid residues; X.sup.5 is
a covalent bond or a spacer domain, the spacer domain comprising at
least one amino acid or a combination of multiple or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X).sub.m--, wherein m is 1 to 6 and X is selected from the
group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X.sup.9
is a covalent bond or an aspartate residue; and n is 0, 1 or 2.
15. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sup.1 or Sp.sup.2 is a spacer comprising at least one
amino acid residue, said spacer comprising an antigenic domain,
wherein the antigenic domain comprises the sequence TABLE-US-00002
(SEQ ID NO: 40)
X.sup.20-(D-Y-K-X.sup.2-X.sup.3-D).sub.n-X.sup.5-(D-Y-K-X.sup.7-X.sup.8-D-
-X.sup.9-K)-X.sup.21
where: D, Y, K are their representative amino acids; X.sup.20 and
X.sup.21 are independently a hydrogen or a covalent bond; each
X.sup.2, X.sup.3, X.sup.7 and X.sup.8 is independently an amino
acid residue selected from the group consisting of aromatic amino
acid residues and hydrophilic amino acid residues; X.sup.5 is a
covalent bond or a spacer domain, the spacer domain comprising at
least one amino acid or a combination of multiple or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X).sub.m--, wherein m is 1 to 6 and X is selected from the
group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X.sup.9
is a covalent bond or an aspartate residue; and n is at least
2.
16. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sup.1 or Sp.sup.2 is a spacer comprising at least one
amino acid residue, said spacer comprising an antigenic domain,
wherein the antigenic domain comprises the sequence TABLE-US-00003
(SEQ ID NO: 45)
X.sup.20-X.sup.10-(D-Y-K-X.sup.2-X.sup.3-D).sub.n-X.sup.5-(D-Y-K-X.sup.7-X-
.sup.8-D-X.sup.9-K)-X.sup.21
where: D, Y, and K are their representative amino acids; X.sup.20
and X.sup.21 are independently a hydrogen or a covalent bond;
X.sup.10 is a covalent bond or an amino acid; each X.sup.2,
X.sup.3, X.sup.7 and X.sup.8 is independently an amino acid residue
selected from the group consisting of aromatic amino acid residues
and hydrophilic amino acid residues; X.sup.5 is a covalent bond or
a spacer domain, the spacer domain comprising at least one amino
acid or a combination of multiple or alternating histidine
residues, said combination comprising His-Gly-His, or
-(His-X).sub.m--, wherein m is 1 to 6 and X is selected from the
group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; X.sup.9
is a covalent bond or an aspartate residue; and n is at least
2.
17. The polypeptide, protein or protein fragment of claim 1,
wherein Sp.sup.1 or Sp.sup.2 is a spacer comprising at least one
amino acid residue, said spacer comprising an antigenic domain,
wherein the antigenic domain comprises the sequence TABLE-US-00004
(SEQ ID NO: 42)
X.sup.20-(D-X.sup.11-Y-X.sup.12-X.sup.13)n-X.sup.14-(D-X.sup.11-Y-X.sup.12-
-X.sup.13-D-X.sup.15-K)-X.sup.21
where: D, Y and K are their representative amino acids; X.sup.20
and X.sup.21 are independently a hydrogen or a covalent bond; each
X.sup.11 is a covalent bond or an amino acid; each X.sup.12 is an
amino acid selected from the group consisting of aromatic amino
acid residues and hydrophilic amino acid residues; each X.sup.13 is
a covalent bond or at least one amino acid selected from the group
consisting of aromatic amino acid residues and hydrophilic amino
acid residues; X.sup.14 is a covalent bond or a spacer domain, the
spacer domain comprising at least one amino acid or alternating
histidine residues, said combination comprising His-Gly-His, or
-(His-X).sub.m--, wherein m is 1 to 6 and X is selected from the
group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; X.sup.15
is a covalent bond or an aspartate residue; and n is 0 or at least
1.
18. A polypeptide, protein or protein fragment represented by the
formula
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-His).sub.t-Sp.sub.2-R.s-
ub.2, wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24)
is a metal ion-affinity peptide, t is at least 2, R.sub.1 is
hydrogen, a polypeptide, protein or protein fragment, Sp.sub.1 is a
covalent bond or a spacer comprising at least one amino acid
residue, R.sub.2 is hydrogen, a polypeptide, protein or protein
fragment, Sp.sub.2 is a covalent bond or a spacer comprising at
least one amino acid residue, Z.sub.1 is an amino acid residue
selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu,
Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z.sub.2 is an amino
acid residue selected from the group consisting of Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr,
and Val.
19. The peptide of claim 18, wherein Z.sub.1 and Z.sub.2 are
selected from the group consisting of: (a) Z.sub.1 is Asn and
Z.sub.2 is Lys; and (b) Z.sub.1 is Lys and Z.sub.2 is Gly.
20. A polypeptide, protein or protein fragment represented by the
formula
R.sub.1-Sp.sub.1-[(His-Z.sub.1-His-Arg-His-Z.sub.2-His)-Sp.sub.2].sub.t-R-
.sub.2, wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO:
24) is a metal ion-affinity peptide, t is at least 2, R.sub.1 is
hydrogen, a polypeptide, protein or protein fragment, Sp.sub.1 is a
covalent bond or a spacer comprising at least one amino acid
residue, R.sub.2 is hydrogen, a polypeptide, protein or protein
fragment, Sp.sub.2 is a covalent bond or a spacer comprising at
least one amino acid residue, Z.sub.1 is an amino acid residue
selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu,
Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val, and Z.sub.2 is an amino
acid residue selected from the group consisting of Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr,
and Val; and each Sp.sub.2 of the recombinant polypeptides,
proteins or protein fragments may be the same or different.
21. The peptide of claim 20, wherein Z.sub.1 and Z.sub.2 are
selected from the group consisting of: (a) Z.sub.1 is Asn and
Z.sub.2 is Lys; and (b) Z.sub.1 is Lys and Z.sub.2 is Gly.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
10/460,524, filed Jun. 12, 2003, which is a non-provisional
application claiming priority from provisional Application Ser. No.
60/388,059, filed Jun. 12, 2002, the content of each of which is
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to affinity peptides, fusion proteins
containing affinity peptides, genes coding for such proteins,
expression vectors and transformed microorganisms containing such
genes, and methods for the purification of the fusion proteins.
BACKGROUND OF THE INVENTION
[0003] The possibility of preparing hybrid genes by gene technology
has opened up new routes for the analysis of recombinant proteins.
By linking the coding gene sequence of a desired protein to the
coding gene sequence of a protein fragment having a high affinity
for a ligand (affinity peptide), it is possible to purify desired
recombinant proteins in the form of fusion proteins in one-step
using the affinity peptide.
[0004] Immobilized metal affinity chromatography (IMAC), also known
as metal chelate affinity chromatography (MCAC), is a specialized
aspect of affinity chromatography. The principle behind IMAC lies
in the fact that many transition metal ions, e.g., nickel, zinc and
copper, can coordinate to the amino acids histidine, cysteine, and
tryptophan via electron donor groups on the amino acid side chains.
To utilize this interaction for chromatographic purposes, the metal
ion is typically immobilized onto an insoluble support. This can be
done by attaching a chelating group to the chromatographic matrix.
Most importantly, to be useful, the metal of choice must have a
higher affinity for the matrix than for the compounds to be
purified.
[0005] In U.S. Pat. No. 4,569,794, Smith et al. disclose the
preparation of a fusion protein containing a metal ion-affinity
peptide linker and a biologically active polypeptide, expressing
the fusion protein, and purifying it using immobilized metal ion
chromatography. Because essentially any biologically active
polypeptide could be used, this approach enabled the convenient
expression and purification of essentially biologically active
polypeptide by immobilized metal ion chromatography.
[0006] In U.S. Pat. Nos. 5,310,663 and 5,284,933, Dobeli et al.
disclose a process for separating a biologically active polypeptide
from impurities by producing the desired polypeptide as a fusion
protein containing a metal ion-affinity peptide linker comprising 2
to 6 adjacent histidine residues. Although Dobeli et al.'s metal
ion-affinity peptide provides greater metal affinity relative to
certain of the sequences disclosed by Smith et al., there is some
cautionary evidence that proteins containing His-tags may differ
from their wild-type counterparts in dimerization/oligomerization
properties. For example, Wu and Filutowicz present evidence that
the biochemical properties of the pi(30.5) protein of plasmid R6K,
a DNA binding protein, were fundamentally altered due to the
presence of an N-terminal 6.times. His-tag. Wu, J. and Filutowicz,
M., Acta Biochim. Pol., 46:591-599, 1999. In addition,
Rodriguez-Viciana et al. stated that V12 Ras proteins expressed as
histidine-tagged fusion proteins exhibited poor biological
activity. Rodriguez-Viciana, P., et al., Cell, 89:457-67, 1997.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention is a peptide which is
relatively hydrophilic, is capable of exhibiting appropriate
biological activity, and has a relatively high affinity for
coordinating metals. Advantageously, this metal ion-affinity
peptide may be incorporated into a fusion protein to enable ready
purification of the fusion protein from aqueous solutions by
immobilized metal affinity chromatography. In addition to the metal
ion-affinity peptide, the fusion protein typically comprises a
protein or polypeptide of interest, covalently linked, directly or
indirectly, to the metal ion-affinity peptide.
[0008] Briefly, therefore, the present invention is directed to a
peptide represented by the formula
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-His)-Sp.sub.2-R.sub.2,
wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide, R.sub.1 is hydrogen, a polypeptide,
protein or protein fragment, Sp.sub.1 is a covalent bond or a
spacer comprising at least one amino acid residue, R.sub.2 is
hydrogen, a polypeptide, protein or protein fragment, Sp.sub.2 is a
covalent bond or a spacer comprising at least one amino acid
residue, Z.sub.1 is an amino acid residue selected from the group
consisting of Ala, Arg, Asn, Asp, Gln, Glu, Ile, Lys, Phe, Pro,
Ser, Thr, Trp, and Val; and Z.sub.2 is an amino acid residue
selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr, and Val.
[0009] The present invention is further directed to a process for
separating a recombinant protein or polypeptide from a liquid
mixture wherein the recombinant protein or polypeptide comprises a
metal ion-affinity peptide having the sequence
His-Z.sub.1-His-Arg-His-Z.sub.2-His (SEQ ID NO: 24) and Z.sub.1 and
Z.sub.2 are as previously defined. In the process, the mixture is
combined with a solid support having immobilized metal ions to bind
the recombinant protein or polypeptide, and eluting the fusion
protein from the solid support.
[0010] The present invention is further directed to vectors and
host cells for recombinant expression of the nucleic acid molecules
described herein, as well as methods of making such vectors and
host cells and for using them for production of the polypeptides or
peptides of the present invention by recombinant techniques.
[0011] The present invention is further directed to a kit for the
expression and/or separation of the recombinant proteins or
polypeptides from a mixture wherein the recombinant proteins or
polypeptides contain the sequence
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-His)-Sp.sub.2-R.sub.2,
and R.sub.1, R.sub.2, Sp.sub.1, Sp.sub.2, Z.sub.1 and Z.sub.2 are
as previously defined. The kit may comprise, in separate
containers, the nucleic acid components to be assembled into a
vector encoding for a fusion protein comprising a protein or
polypeptide covalently operably linked directly or indirectly to an
immobilized metal ion-affinity peptide. In addition, or
alternatively, the kit may be comprised of one or more of the
following: buffers, enzymes, a chromatography column comprising a
resin containing immobilized metal ions and an instructional
brochure explaining how to use the kit.
[0012] Other objects and advantages of the present invention will
become apparent as the detailed description of the invention
proceeds.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013] The present invention generally relates to the expression
and purification of recombinant polypeptides, proteins or protein
fragments containing a metal ion-affinity peptide. In addition to
the metal ion-affinity peptide, the recombinant polypeptides and
proteins will typically also contain a target polypeptide, protein
or fragment thereof covalently linked to the metal ion-affinity
peptide. In one embodiment, the target polypeptide, protein or
protein fragment is a biologically active protein or protein
fragment. Advantageously, the metal ion-affinity peptide enables
the recombinant polypeptides and proteins to be readily purified
from a liquid sample by means of metal ion affinity
chromatography.
[0014] The fusion proteins of this invention are prepared by
recombinant DNA methodology. In accordance with the present
invention, a gene sequence coding for a desired protein is
isolated, synthesized or otherwise obtained and operably linked to
a DNA sequence coding for the metal ion-affinity peptide. The
hybrid gene containing the gene for a desired protein operably
linked to a DNA sequence encoding the metal ion-affinity peptide is
referred to as a chimeric gene.
[0015] In one embodiment, the metal ion-affinity peptide is
covalently linked to the carboxy terminus of the target
polypeptide, protein or protein fragment. In another embodiment,
the metal ion-affinity peptide is covalently linked to the amino
terminus of the target polypeptide, protein or protein fragment. In
each of these embodiments, the metal ion-affinity peptide and the
target polypeptide, protein or protein fragment may be directly
attached by means of a peptide bond or, alternatively, the two may
be separated by a linker. When present, the linker may provide
other functionality to the recombinant polypeptide, protein or
protein fragment.
[0016] The recombinant polypeptides, proteins or protein fragments
of the present invention are defined by the general formula
(I):
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-H
is)-Sp.sub.2-R.sub.2 (I)
wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide; Z.sub.1 is an amino acid residue
selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu,
Ile, Lys, Phe, Pro, Ser, Thr, Trp, and Val; and Z.sub.2 is an amino
acid residue selected from the group consisting of Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Tyr and
Val. In addition, R.sub.1 is hydrogen, a polypeptide, protein or
protein fragment, Sp.sub.1 is a covalent bond or a spacer
comprising at least one amino acid residue, R.sub.2 is hydrogen, a
polypeptide, protein or protein fragment, Sp.sub.2 is a covalent
bond or a spacer comprising at least one amino acid residue. Thus,
for example, R.sub.1 or R.sub.2 may comprise a target polypeptide,
protein, or protein fragment which is directly or indirectly linked
to the metal ion-affinity peptide.
[0017] Metal Ion-Affinity Peptide
[0018] In one embodiment, the recombinant polypeptide, protein or
protein fragment is defined by formula (I), wherein Z.sub.1 is an
amino acid selected from the group consisting of Ala, Asn, Ile,
Lys, Phe, Ser, Thr, and Val; and Z.sub.2 is an amino acid selected
from the group consisting of Ala, Asn, Gly, Lys, Ser, Thr, Tyr; and
R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2 are as previously defined.
Thus, for example, in this embodiment the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2) may be at the
carboxy or amino terminus of the metal ion-affinity polypeptide. In
addition, the target polypeptide, protein or protein fragment
(R.sub.1 or R.sub.2), may be directly fused (when Sp.sub.1 or
Sp.sub.2 is a covalent bond) or separated from the metal
ion-affinity polypeptide by a spacer (when Sp.sub.1 or Sp.sub.2 is
one or more amino acid residues) regardless of whether the target
polypeptide, protein or protein fragment is fused to the amino or
carboxy terminus of the metal ion-affinity polypeptide.
[0019] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
an amino acid selected from the group consisting of Asn and Lys;
and Z.sub.2 is an amino acid selected from the group consisting of
Gly and Lys; and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2 are as
previously defined. For example, in one such embodiment, the
recombinant polypeptide, protein or protein fragment is defined by
formula (I) wherein Z.sub.1 is Asn, Z.sub.2 is Lys and R.sub.1,
R.sub.2 Sp.sub.1, and Sp.sub.2 are as previously defined. By way of
further example, in another such embodiment, the recombinant
polypeptide, protein or protein fragment is defined by formula (I)
wherein Z.sub.1 is Lys and Z.sub.2 is Gly. In each of these
alternatives, the target polypeptide, protein or protein fragment
(R.sub.1 or R.sub.2) may be at the carboxy or amino terminus of the
metal ion-affinity polypeptide. In addition, the target
polypeptide, protein or protein fragment (R.sub.1 or R.sub.2), may
be directly fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or
separated from the metal ion-affinity polypeptide by a spacer (when
Sp.sub.1 or Sp.sub.2 is one or more amino acid residues) regardless
of whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0020] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
Ile, Z.sub.2 is Asn, and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2
are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R.sub.1 or
R.sub.2) may be at the carboxy or amino terminus of the metal
ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2), may be directly
fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or separated
from the metal ion-affinity polypeptide by a spacer (when Sp.sub.1
or Sp.sub.2 is one or more amino acid residues) regardless of
whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0021] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
Thr, Z.sub.2 is Ser, and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2
are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R.sub.1 or
R.sub.2) may be at the carboxy or amino terminus of the metal
ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2), may be directly
fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or separated
from the metal ion-affinity polypeptide by a spacer (when Sp.sub.1
or Sp.sub.2 is one or more amino acid residues) regardless of
whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0022] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
Ser, Z.sub.2 is Tyr, and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2
are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R.sub.1 or
R.sub.2) may be at the carboxy or amino terminus of the metal
ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2), may be directly
fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or separated
from the metal ion-affinity polypeptide by a spacer (when Sp.sub.1
or Sp.sub.2 is one or more amino acid residues) regardless of
whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0023] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
Val, Z.sub.2 is Ala, and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2
are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R.sub.1 or
R.sub.2) may be at the carboxy or amino terminus of the metal
ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2), may be directly
fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or separated
from the metal ion-affinity polypeptide by a spacer (when Sp.sub.1
or Sp.sub.2 is one or more amino acid residues) regardless of
whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0024] In another embodiment, the recombinant polypeptide, protein
or protein fragment is defined by formula (I), wherein Z.sub.1 is
Ala, Z.sub.2 is Lys, and R.sub.1, R.sub.2, Sp.sub.1, and Sp.sub.2
are as previously defined. Thus, for example, in this embodiment
the target polypeptide, protein or protein fragment (R.sub.1 or
R.sub.2) may be at the carboxy or amino terminus of the metal
ion-affinity polypeptide. In addition, the target polypeptide,
protein or protein fragment (R.sub.1 or R.sub.2), may be directly
fused (when Sp.sub.1 or Sp.sub.2 is a covalent bond) or separated
from the metal ion-affinity polypeptide by a spacer (when Sp.sub.1
or Sp.sub.2 is one or more amino acid residues) regardless of
whether the target polypeptide, protein or protein fragment is
fused to the amino or carboxy terminus of the metal ion-affinity
polypeptide.
[0025] In a further embodiment, R.sub.1 may be a polypeptide which
drives expression of the fusion protein and R.sub.2 is the target
polypeptide, protein or protein fragment. In this embodiment, each
of Sp.sub.1 and Sp.sub.2 may be a covalent bond or a spacer,
independently of the other. Thus, for example, R.sub.1 may be
directly fused to the metal ion-affinity peptide or separated from
the metal ion-affinity peptide by a spacer independently of whether
R.sub.2 is directly fused to the metal ion-affinity peptide or
separated from the metal ion-affinity peptide by a spacer; all of
these combinations and permutations are contemplated. This type of
arrangement is particularly useful when chimeric proteins are
constructed which comprise epitopes from two portions of antigenic
protein or from two different antigenic proteins. Such chimeric
proteins may be useful in vaccine preparations.
[0026] In another embodiment, the recombinant polypeptides,
proteins or protein fragments of the present invention comprise
multiple copies of the metal ion-affinity peptide
(His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) wherein
Z.sub.1 and Z.sub.2 are as previously defined. In this embodiment,
the additional copies of the metal affinity peptide may occur in
either or both of the spacer domains (Sp.sub.1 and Sp.sub.2) or in
either or both of the other domains (R.sub.1 and R.sub.2) of the
recombinant polypeptides, proteins or protein fragments. Thus, for
example, in one embodiment a second copy of the metal ion-affinity
peptide (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24)
wherein Z.sub.1 and Z.sub.2 are as previously defined is located in
one of the spacer domains (Sp.sub.1 or Sp.sub.2) or other domains
(R.sub.1 and R.sub.2) of the recombinant polypeptides, proteins or
protein fragments. By way of further example, in another embodiment
two additional copies of the metal ion-affinity peptide
(His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) wherein
Z.sub.1 and Z.sub.2 are as previously defined are located in the
spacer domains (Sp.sub.1 or Sp.sub.2) or other domains (R.sub.1 and
R.sub.2) of the recombinant polypeptides, proteins or protein
fragments. By way of further example, in another embodiment at
least three additional copies of the metal ion-affinity peptide
(His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) wherein
Z.sub.1 and Z.sub.2 are as previously defined are located in the
spacer domains (Sp.sub.1 or Sp.sub.2) or other domains (R.sub.1 and
R.sub.2) of the recombinant polypeptides, proteins or protein
fragments. In each of these embodiments, the multiple copies of the
metal ion-affinity peptide may be separated by one or more amino
acid residues (i.e., a spacer) as described herein. Alternatively,
in each of these embodiments the multiple copies of the metal
ion-affinity peptide may be directly linked to each other without
any intervening amino acid residues. Thus, for example, in one such
embodiment the recombinant polypeptides, proteins or protein
fragments of the present invention may be defined by the general
formula (II):
R.sub.1-Sp.sub.1-(His-Z.sub.1-His-Arg-His-Z.sub.2-His).sub.t-Sp.sub.2-R.-
sub.2 (II)
wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide; t is at least 2 and R.sub.1, R.sub.2,
Z.sub.1, Z.sub.2, Sp.sub.1 and Sp.sub.2 are as previously defined.
By way of further example, in one such embodiment the recombinant
polypeptides, proteins or protein fragments of the present
invention may be defined by the general formula (III):
R.sub.1-Sp.sub.1-[(His-Z.sub.1-His-Arg-His-Z.sub.2-His)-Sp.sub.2].sub.t--
R.sub.2 (III)
wherein (His-Z.sub.1-His-Arg-His-Z.sub.2-His) (SEQ ID NO: 24) is a
metal ion-affinity peptide; t is at least 2 and R.sub.1, R.sub.2,
Z.sub.1, Z.sub.2, Sp.sub.1 and Sp.sub.2 are as previously defined;
in addition, each Sp.sub.2 of the recombinant polypeptides,
proteins or protein fragments corresponding to general formula
(III) may be the same or different.
[0027] Target Polypeptide, Protein or Protein Fragment
[0028] The target polypeptide, protein or protein fragment may be
composed of any proteinaceous substance that can be expressed in
transformed host cells. Accordingly, the present invention may be
beneficially employed to produce substantially any prokaryotic or
eukaryotic, simple or conjugated, protein that can be expressed by
a vector in a transformed host cell. For example, the target
protein may be [0029] a) an enzyme, whether oxidoreductase,
transferase, hydrolase, lyase, isomerase or ligase; [0030] b) a
storage protein, such as ferritin or ovalbumin or a transport
protein, such as hemoglobin, serum albumin or ceruloplasmin; [0031]
c) a protein that functions in contractile and motile systems such
as actin or myosin; [0032] d) any of a class of proteins that serve
a protective or defense function, such as the blood protein
fibrinogen or a binding protein, such as antibodies or
immunoglobulins that bind to and thus neutralize antigens; [0033]
e) a hormone such as human Growth Hormone, somatostatin, prolactin,
estrone, progesterone, melanocyte, thyrotropin, calcitonin,
gonadotropin and insulin; [0034] f) a hormone involved in the
immune system, such as interleukin-1, interleukin-2, colony
stimulating factor, macrophage-activating factor and interferon;
[0035] g) a toxic protein, such as ricin from castor bean or
gossypin from cotton linseed; [0036] h) a protein that serves as
structural elements such as collagen, elastin, alpha-keratin,
glyco-proteins, viral proteins and muco-proteins; or [0037] i) a
synthetic protein, defined generally as any sequence of amino acids
not occurring in nature. In general, the target polypeptide,
protein or protein fragment may be a constituent of the R.sub.1 and
R.sub.2 moieties of the recombinant polypeptides, proteins or
protein fragments corresponding to general formulae (I), (II) and
(III).
[0038] Genes coding for the various types of protein molecules
identified above may be obtained from a variety of prokaryotic or
eukaryotic sources, such as plant or animal cells or bacteria
cells. The genes can be isolated from the chromosome material of
these cells or from plasmids of prokaryotic cells by employing
standard, well-known techniques. A variety of naturally occurring
and synthesized plasmids having genes coding for many different
protein molecules are not commercially available from a variety of
sources. The desired DNA also can be produced from mRNA by using
the enzyme reverse transcriptase. This enzyme permits the synthesis
of DNA from an RNA template.
[0039] In one embodiment, R.sub.1 may be a protein which enhances
expression and R.sub.2 is the target polypeptide, protein, or
protein fragment. It is well known that the presence of some
proteins in a cell result in expression of genes. If a chimeric
protein contains an active portion of the protein which prompts or
enhances expression of the gene encoding it, greater quantities of
the protein may be expressed than if it were not present.
[0040] Linker and Other Optional Elements
[0041] In one embodiment, the recombinant polypeptide, protein or
protein fragment includes a spacer (Sp.sub.1 or Sp.sub.2) between
the metal ion-affinity polypeptide and the target polypeptide,
protein or protein fragment. If present, the spacer may simply
comprise one or more, e.g., three to ten amino acid residues,
separating the metal ion-affinity peptide from the target
polypeptide, protein or protein fragment. Alternatively, the spacer
may comprise a sequence which imparts other functionality, such as
a proteolytic cleavage site, a fusion protein, a secretion sequence
(e.g. OmpA or OmpT for E. coli, preprotrypsin for mammalian cells,
a-factor for yeast, and melittin for insect cells), a leader
sequence for cellular targeting, antibody epitopes, or IRES
(internal ribosomal entry sequences) sequences.
[0042] In one embodiment, the spacer is selected from among
hydrophilic amino acids to increase the hydrophilic character of
the recombinant polypeptide, protein or protein fragment.
Alternatively, the amino acid(s) of the spacer domain may be
selected to impart a desired folding to the recombinant
polypeptide, protein or protein fragment thereby increasing
accessability to one or more regions of the molecule. For example,
the spacer domain may comprise glycine residues which results in a
protein folding conformation which allows for improved
accessibility to antibodies.
[0043] In another embodiment, the spacer comprises a cleavage site
which consists of a unique amino acid sequence cleavable by use of
a sequence-specific proteolytic agent. Such a site would enable the
metal ion-affinity polypeptide to be readily cleaved from the
target polypeptide, protein or protein fragment by digestion with a
proteolytic agent specific for the amino acids of the cleavage
site. Alternatively, the metal ion-affinity peptide may be removed
from the desired protein by chemical cleavage using methods known
to the art.
[0044] When present, the cleavable site may be located at the amino
or carboxy terminus of the target peptide. Preferably, the
cleavable site is immediately adjacent the desired protein to
enable separation of the desired protein from the metal
ion-affinity peptide. This cleavable site preferably does not
appear in the desired protein. In one embodiment, the cleavable
site is located at the amino terminus of the desired protein. If
the cleavable site is located at the amino terminus of the desired
protein and if there are remaining extraneous amino acids on the
desired protein after cleavage with the proteolytic agent, an
endopeptidase such as trypsin, clostropain or furin may be utilized
to remove these remaining amino acids, thus resulting in a highly
purified desired protein. Further examples of proteolytic enzymatic
agents useful for cleavage are papain, pepsin, plasmin, thrombin,
enterokinase, and the like. Each effects cleavage at a particular
amino acid sequence which it recognizes.
[0045] Digestion with a proteolytic agent may occur while the
fusion protein is still bound to the affinity resin or
alternatively, the fusion protein may be eluted from the affinity
resin and then digested with the proteolytic agent in order to
further purify the desired protein. Preferably, the amino acid
sequence of the proteolytic cleavage site is unique, thus
minimizing the possibility that the proteolytic agent will cleave
the desired protein. In one embodiment, the cleavable site
comprises amino acids for an enterokinase, thrombin or a Factor Xa
cleavage site.
[0046] Enterokinase recognizes several sequences: Asp-Lys;
Asp-Asp-Lys; Asp-Asp-Asp-Lys (SEQ ID NO: 25); and
Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 26). The only known natural
occurrence of Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 26) is in the protein
trypsinogen which is a natural substrate for bovine enterokinase
and some yeast proteins. As such, by interposing a fragment
containing the amino acid sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO:
26) as a cleavable site between the metal ion-affinity polypeptide
and the amino terminus of the target polypeptide, protein or
protein fragment, the metal ion-affinity polypeptide can be
liberated from the desired protein by use of bovine enterokinase
with very little likelihood that this enzyme will cleave any
portion of the desired protein itself.
[0047] Thrombin cleaves on the carboxy-terminal side of arginine in
the following sequence: Leu-Val-Pro-Arg-Gly-X (SEQ ID NO: 27),
where X is a non-acidic amino acid. Factor Xa protease (i.e., the
activated form of Factor X) cleaves after the Arg in the following
sequences: Ile-Glu-Gly-Arg-X (SEQ ID NO: 28), Ile-Asp-Gly-Arg-X
(SEQ ID NO: 29), and Ala-Glu-Gly-Arg-X (SEQ ID NO: 30), where X is
any amino acid except proline or arginine. A fusion protein
comprising the 31 amino-terminal residues of the cII protein, a
Factor Xa cleavage site and human .beta.-globin was shown to be
cleaved by Factor Xa and generate authentic .beta.-globin. A
limitation of the Factor Xa-based fusion systems is the fact that
Factor Xa has been reported to cleave at arginine residues that are
not present within in the Factor Xa recognition sequence.
Lauritzen, C. et al., Protein Expr. and Purif., 5-6:372-378
(1991).
[0048] While less preferred, other unique amino acid sequences for
other cleavable sites may also be employed in the spacer without
departing from the spirit or scope of the present invention. For
instance, the spacer may be composed, at least in part, of a pair
of basic amino acids, i.e., Arg, His or Lys. This sequence is
cleaved by kallikreins, a glandular enzyme. Also, the spacer may be
composed, at least in part, of Arg-Gly, since it is known that the
enzyme thrombin will cleave after the Arg if this residue is
followed by Gly.
[0049] Regardless of whether a cleavage site is present, the
recombinant polypeptide, protein or protein fragment may comprise
an antigenic domain in a spacer region (Sp.sub.1 or Sp.sub.2). For
example, in one embodiment of the present invention, the
recombinant polypeptide, protein or protein fragment comprises one
or multiple copies of an antigenic domain generally corresponding
to the FLAG.RTM. (Sigma-Aldrich, St. Louis, Mo.) peptide sequence
joined to a linking sequence containing a single enterokinase
cleavage site. Such antigenic domains generally correspond to the
sequence:
[0050]
X.sup.20--(X.sup.1--Y--K--X.sup.2--X.sup.3-D-X.sup.4).sub.n--X.sup.-
5--(X.sup.1--Y--K--X.sup.7--X.sup.8-D-X.sup.9--K)--X.sup.21 (SEQ ID
NO: 39)
[0051] where:
[0052] D, Y and K are their representative amino acids;
[0053] X.sup.20 and X.sup.21 are independently a hydrogen or a
covalent bond;
[0054] each X.sup.1 and X.sup.4 is independently a covalent bond or
at least one amino acid residue, if other than a covalent bond,
preferably at least one amino acid residue selected from the group
consisting of aromatic amino acid residues and hydrophilic amino
acid residues, more preferably at least one hydrophilic amino acid
residue, and still more preferably at least one an aspartate
residue;
[0055] each X.sup.2, X.sup.3, X.sup.7 and X.sup.8 is independently
an amino acid residue, preferably an amino acid residue selected
from the group consisting of aromatic amino acid residues and
hydrophilic amino acid residues, more preferably a hydrophilic
amino acid residue, and still more preferably an aspartate
residue;
[0056] X.sup.5 is a covalent bond or a spacer domain comprising at
least one amino acid, if other than a covalent bond, preferably a
histidine residue, a glycine residue or a combination of multiple
or alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X).sub.m--, wherein m is 1 to 6 and X is
selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and
Val;
[0057] X.sup.9 is a covalent bond or D; and
[0058] n is 0, 1 or 2.
[0059] In this embodiment, the amino acid sequence
X.sup.20--(X.sup.1--Y--K--X.sup.2--X.sup.3-D-X.sup.4).sub.n (SEQ ID
NO: 35) comprises an antigenic domain
--X.sup.1--Y--K--X.sup.2--X.sup.3-D- (SEQ ID NO: 36) joined in
tandem which are joined to a linking sequence
(X.sup.1--Y--K--X.sup.7--X.sup.8-D-X.sup.9--K) (SEQ ID NO: 37). The
antigenic domains may be immediately adjacent to each other when n
is at least one and X.sup.4 is a covalent bond; optionally, X.sup.4
may be a spacer domain interposed between the multiple copies of
antigenic domains. The linking sequence contains a single
enterokinase cleavable site which is represented by the sequence
--X.sup.7--X.sup.8-D-X.sup.9--K (SEQ ID NO: 38), where X.sup.7 and
X.sup.8 may be an amino acid residue or a covalent bond and X.sup.9
is a covalent bond or an aspartate residue. In one embodiment, each
X.sup.7, X.sup.8 and X.sup.9 is independently an aspartate residue
thus resulting in the enterokinase cleavable site DDDDK (SEQ ID NO:
26) which is preferably located immediately adjacent to the amino
terminus of the target peptide. When n is at least one and X.sup.5
is a covalent bond, the multiple copies of antigenic domains may be
immediately adjacent to the linking sequence; optionally, X.sup.5
may be a spacer domain interposed between the linking sequence and
the antigenic domains. When each X.sup.4 and X.sup.5 is
independently a spacer domain, it is preferred that the amino acid
residue(s) of each X.sup.4 and X.sup.5 impart one or more desired
properties to the antigenic domain; for example, the amino acids of
the spacer domain may be selected to impart a desired folding to
the identification polypeptide thereby increasing accessibility to
the antibody. In another embodiment, the amino acids of the spacer
domain X.sup.4 and X.sup.5 may be selected to impart a desired
affinity characteristic such as a combination of multiple or
alternating histidine residues capable of chelating to an
immobilized metal ion on a resin or other matrix. Furthermore,
these desired properties may be designed into other areas of the
identification polypeptide; for example, the amino acids
represented by X.sup.2 and X.sup.3 may be selected to impart a
desired peptide folding or a desired affinity characteristic for
use in affinity purification.
[0060] In another embodiment, the spacer comprises multiple copies
of an antigenic domain. For example, in one embodiment the spacer
may comprise a linking sequence containing a single enterokinase or
other cleavage site, or generally correspond to the sequence:
[0061]
X.sup.20-(D-Y--K--X.sup.2--X.sup.3-D).sub.n-X.sup.5-(D-Y--K--X.sup.-
7--X.sup.8-D-X.sup.9--K)--X.sup.21 (SEQ ID NO: 40)
[0062] where:
[0063] D, Y, K are their representative amino acids;
[0064] X.sup.20 and X.sup.21 are independently a hydrogen or a
covalent bond; each X.sup.2, X.sup.3, X.sup.7 and X.sup.8 is
independently an amino acid residue, preferably an amino acid
residue selected from the group consisting of aromatic amino acid
residues and hydrophilic amino acid residues, more preferably a
hydrophilic amino acid residue, and still more preferably an
aspartate residue;
[0065] X.sup.5 is a covalent bond or a spacer domain comprising at
least one amino acid, if other than a covalent bond, preferably a
histidine residue, a glycine residue or a combination of multiple
or alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X).sub.m--, wherein m is 1 to 6 and X is
selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and
Val;
[0066] X.sup.9 is a covalent bond or an aspartate residue; and
[0067] n is at least 2.
[0068] In this embodiment, the amino acid sequence
X.sup.20-(D-Y--K--X.sup.2--X.sup.3-D).sub.n (SEQ ID NO: 41)
represents the multiple copies of the antigenic domain
D-Y--K--X.sup.2--X.sup.3-D (SEQ ID NO: 31) in tandem which are
joined to a linking sequence
(D-Y--K--X.sup.7--X.sup.8-D-X.sup.9--K) (SEQ ID NO: 32). In this
embodiment, one antigenic domain is immediately adjacent to another
antigenic domain, i.e., no intervening spacer domains, and the
multiple copies of the antigenic domain are immediately adjacent to
the linking sequence when X.sup.5 is a covalent bond. The linking
sequence contains a single enterokinase cleavable site which is
represented by the sequence --X.sup.7--X.sup.8-D-X.sup.9-K (SEQ ID
NO: 38), where X.sup.7 and X.sup.8 may be a covalent bond or an
amino acid residue, preferably an aspartate residue, and X.sup.9 is
a covalent bond or an aspartate residue. In one embodiment, each
X.sup.7, X.sup.8 and X.sup.9 is independently an aspartate residue
thus resulting in the enterokinase cleavable site DDDDK (SEQ ID NO:
26) which is preferably adjacent to the amino terminus of the
target peptide. Optionally, the multiple copies of the antigenic
domain are joined to the linking sequence by a spacer X.sup.5 when
X.sup.5 is at least one amino acid residue. When X.sup.5 is a
spacer domain, it is preferred that the amino acid residue(s) of
X.sup.5 impart one or more desired properties to the recombinant
polypeptide, protein or protein fragment; for example, the amino
acids of the spacer domain may be selected to impart a desired
folding to the recombinant polypeptide, protein or protein fragment
thereby increasing accessibility to the antibody. In another
embodiment, the amino acids of the spacer domain may be selected to
impart a desired affinity characteristic such as a combination of
multiple or alternating histidine residues capable of chelating to
an immobilized metal ion on a resin or other matrix. Furthermore,
these desired properties may be designed into other areas of the
spacer; for example, the amino acids represented by X.sup.2 and
X.sup.3 may be selected to impart a desired peptide folding or a
desired affinity characteristic for use in affinity
purification.
[0069] When the affinity polypeptide is located at the amino
terminus of the target polypeptide, protein or protein fragment, it
is often desirable to design the amino acid sequence such that an
initiator methionine is present. Accordingly, in one embodiment of
the present invention, the recombinant polypeptide, protein or
protein fragment comprises multiple copies of an antigenic domain,
a linking sequence containing a single enterokinase cleavage site
and generally corresponds to the sequence:
[0070]
X.sup.20--X.sup.10-(D-Y--K--X.sup.2--X.sup.3-D).sub.n-X.sup.5-(D-Y--
-K--X.sup.7--X.sup.8-D-X.sup.9--K)--X.sup.21 (SEQ ID NO: 45)
[0071] where:
[0072] D, Y, and K are their representative amino acids;
[0073] X.sup.20 and X.sup.21 are independently a hydrogen or a
covalent bond;
[0074] X.sup.10 is a covalent bond or an amino acid, if other than
a covalent bond, preferably a methionine residue;
[0075] each X.sup.2, X.sup.3, X.sup.7 and X.sup.5 is independently
an amino acid residue, preferably an amino acid residue selected
from the group consisting of aromatic amino acid residues and
hydrophilic amino acid residues, more preferably a hydrophilic
amino acid residue, and still more preferably an aspartate
residue;
[0076] X.sup.5 is a covalent bond or a spacer domain comprising at
least one amino acid, if other than a bond, preferably a histidine
residue, a glycine residue or a combination of multiple or
alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X).sub.m--, wherein m is 1 to 6 and X is
selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,
and Val;
[0077] X.sup.9 is a covalent bond or an aspartate residue; and
[0078] n is at least 2.
[0079] In this embodiment, the amino acid sequence
X.sup.20--X.sup.10-(D-Y--K--X.sup.2--X.sup.3-D).sub.n (SEQ ID NO:
44) represents the multiple copies of the antigenic domain
D-Y--K--X.sup.2--X.sup.3-D (SEQ ID NO: 31) in tandem which is
flanked by a linking sequence
(D-Y--K--X.sup.7--X.sup.8-D-X.sup.9--K) (SEQ ID NO: 32) and an
initiator amino acid X.sup.10, preferably methionine. The antigenic
domain D-Y--K--X.sup.2--X.sup.3-D with an initiator methionine is
recognized by the M5.RTM. antibody (Sigma-Aldrich, St. Louis, Mo.).
In this embodiment, one antigenic domain is immediately adjacent to
another antigenic domain, i.e., no intervening spacer domains, and
the multiple copies of the antigenic domain are immediately
adjacent to the linking sequence when X.sup.5 is a covalent bond.
The linking sequence contains an enterokinase cleavable site which
is represented by the amino acid sequence
--X.sup.7--X.sup.8-D-X.sup.9--K (SEQ ID NO: 38), where X.sup.7 and
X.sup.8 may be a covalent bond or an amino acid residue, preferably
an aspartate residue, and X.sup.9 is a covalent bond or an
aspartate residue. In one embodiment, each X.sup.7, X.sup.8 and
X.sup.9 is independently an aspartate residue thus resulting in the
enterokinase cleavable site DDDDK (SEQ ID NO: 26) which is
preferably adjacent to the amino terminus of the target peptide.
Optionally, the multiple copies of the antigenic domain are joined
to the linking sequence by a spacer domain X.sup.5 whenX.sup.5 is
at least one amino acid residue. When X.sup.5 is a spacer domain,
it is preferred that the amino acid residue(s) of X.sup.5 impart
one or more desired properties to the affinity polypeptide; for
example, the amino acids of the spacer domain may be selected to
impart a desired folding to the recombinant polypeptide, protein or
protein fragment thereby increasing accessibility to the antibody.
In another embodiment, the amino acids of the spacer domain may be
selected to impart a desired affinity characteristic such as a
combination of multiple or alternating histidine residues capable
of chelating to an immobilized metal ion on a resin or other
matrix. Furthermore, these desired properties may be designed into
other areas of the affinity polypeptide; for example, the amino
acids represented by X.sup.2 and X.sup.3 may be selected to impart
a desired peptide folding or a desired affinity characteristic for
use in affinity purification.
[0080] In another embodiment of the present invention, the
recombinant polypeptide, protein or protein fragment comprises one
or more copies of an antigenic sequence, a linking sequence
containing a single enterokinase cleavable site and generally
corresponds to the sequence:
[0081]
X.sup.20-(D-X.sup.11--Y--X.sup.12--X.sup.13).sub.n--X.sup.14-(D-X.s-
up.11--Y--X.sup.12--X.sup.13-D-X.sup.15--K)--X.sup.21 (SEQ ID NO:
42)
[0082] where:
[0083] D, Y and K are their representative amino acids;
[0084] X.sup.20 and X.sup.21 are independently a hydrogen or a
covalent bond;
[0085] each X.sup.11 is a covalent bond or an amino acid,
preferably Leu;
[0086] each X.sup.12 is an amino acid, preferably selected from the
group consisting of aromatic amino acid residues and hydrophilic
amino acid residues, more preferably a hydrophilic amino acid
residue, and still more preferably an aspartate residue;
[0087] each X.sup.13 is a covalent bond or at least one amino acid,
if other than a covalent bond, preferably selected from the group
consisting of aromatic amino acid residues and hydrophilic amino
acid residues, more preferably a hydrophilic amino acid residue,
and still more preferably an aspartate residue;
[0088] X.sup.14 is a covalent bond or a spacer domain comprising at
least one amino acid, if other than a covalent bond, preferably a
histidine residue, a glycine residue or a combination of multiple
or alternating histidine residues, said combination comprising
His-Gly-His, or -(His-X).sub.m--, wherein m is 1 to 6 and X is
selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and
Val;
[0089] X.sup.15 is a covalent bond or an aspartate residue; and
[0090] n is 0 or at least 1.
[0091] In this embodiment, when n is at least 2, the amino acid
sequence X.sup.20-(D-X.sup.11--Y--X.sup.12--X.sup.13).sub.n (SEQ ID
NO: 43) constitutes multiple copies of the antigenic domain
D-X.sup.11--Y--X.sup.12--X.sup.13 (SEQ ID NO: 33) in tandem which
are joined to a linking sequence
(D-X.sup.11--Y--X.sup.12--X.sup.13-D-X.sup.15--K) (SEQ ID NO: 34).
Additionally, one antigenic domain may be immediately adjacent to
another antigenic domain, i.e., no intervening spacer domains, and
the multiple copies of the antigenic domain may be immediately
adjacent to the linking sequence when X.sup.14 is a covalent bond.
The linking sequence contains a single enterokinase cleavable site
which is represented by the sequence
--X.sup.12--X.sup.13-D-X.sup.15--K, (SEQ ID NO: 38) where X.sup.12
and X.sup.13 may be a covalent bond or an amino acid residue,
preferably an aspartate residue, and X.sup.15 is a covalent bond or
an aspartate residue. In one embodiment, each X.sup.12, X.sup.13
and X.sup.15 is independently an aspartate residue thus resulting
in the enterokinase cleavable site DDDDK (SEQ ID NO: 26) which is
preferably adjacent to the amino terminus of the target peptide.
Optionally, when n is at least two, the multiple copies of the
antigenic domain are joined to the linking sequence by a spacer
X.sup.14 when X.sup.14 is at least one amino acid residue. When
X.sup.14 is a spacer domain, it is preferred that the amino acid
residue(s) of X.sup.14 impart one or more desired properties to the
recombinant polypeptide, protein or protein fragment; for example,
the amino acids of the spacer domain may be selected to impart a
desired folding to the recombinant polypeptide, protein or protein
fragment thereby increasing accessibility to the antibody. In
another embodiment, the amino acids of the spacer domain X.sup.14
may be selected to impart a desired affinity characteristic such as
a combination of multiple or alternating histidine residues capable
of chelating to an immobilized metal ion on a resin or other
matrix.
[0092] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the enzyme glutathione-S-transferase of the
parasite helminth Schistosoma japonicum (SEQ ID NO: 1). The
glutathione-S-transferase may, however, be derived from other
species including human and other mammalian
glutathione-S-transferase. Proteins expressed as fusions with the
enzyme glutathione-S-transferase can be purified under
non-denaturing conditions by affinity chromatography on immobilized
glutathione. Glutathione-agarose beads have a capacity of at least
8 mg fusion protein/ml swollen beads and can be used several times
for different preparations of the same fusion protein. Smith, D. B.
and Johnson, K. S., Gene, 67:31-40, 1988.
[0093] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises a cellulose binding domain (CBD) (SEQ ID NO:
2). CBD's are found in both bacterial and fungal sources and
possess a high affinity for the crystalline form of cellulose. This
property has been useful for purification of fusion proteins using
a cellulose matrix. Fusion proteins have been attached at both the
N- and C-terminus of CBD.
[0094] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the Maltose Binding Protein (MBP) encoded by
the malE gene in E. coli (SEQ ID NO: 3). MBP has found utility in
the formation of chimeric proteins with eukaryotic proteins for
expression in bacterial systems. This system permits expression of
soluble fusion proteins that can readily be purified on immobilized
amylose resin.
[0095] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises Protein A (SEQ ID NO: 4). Protein A is
isolated from Staphylococcus aureus and binds to the Fc origin of
IgG. Fusion proteins containing the IgG binding domains of Protein
A can be affinity purified on IgG resins (e.g., IgG Sepharose 6FF
(Pharmacia Biotech). The signal sequence of Protein A is functional
in E. coli. Fusion proteins using Protein A have shown increased
stability when expressed both in the cytoplasm and periplasm in E.
coli.
[0096] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises Protein G (SEQ ID NO: 5). Protein G is
similar to Protein A with the difference being that Protein G binds
to human serum albumin in addition to IgG. The major disadvantage
is that low pH<3.4 is required to elute the fusion protein.
[0097] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises IgG (SEQ ID NO: 6). Placing the protein of
interest on the C-terminal of IgG generates chimeric proteins. This
allows purification of the fusion protein using either Protein A or
G matrix.
[0098] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the enzyme chloramphenicol acetyl
transferase (CAT) from E. coli (SEQ ID NO: 7). CAT is used in the
form of a C-terminal fusion. CAT is readily translated in E. coli
and allows for over-expression of heterologous proteins. Capture of
fusion proteins is accomplished using a chloramphenicol matrix.
[0099] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises streptavidin (SEQ ID NO: 8). Streptavidin is
used for fusion proteins because of its high affinity and high
specificity for biotin. Streptavidin is a neutral protein, free
from carbohydrates and sulphydryl groups.
[0100] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises b-galactosidase (SEQ ID NO: 9).
b-galactosidase is a enzyme that is utilized as both an N- and
C-terminal fusion protein. Fusion proteins containing
b-galactosidase sequences can be affinity purified on
aminophenyl-b-D-thiogalactosidyl-succinyldiaminohexyl-Sepharose.
However, given that C-terminal fusion proteins are usually
insoluble, the system has limited use in bacterial systems.
N-terminal fusions are soluble in E. coli, but due to the large
size of b-galactosidase, this system is used more often in
eukaryotic gene expression.
[0101] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the Green Fluorescent Protein (GFP) (SEQ ID
NO: 10). GFP is a protein from the jellyfish Aquorea victorea and
many mutant variations of this protein have been used successfully
in most organisms for protein expression. The major use of these
types of fusion proteins is for targeting and determining
physiological function of the host cell protein.
[0102] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises thioredoxin (SEQ ID NO: 11). Thioredoxin is
a relatively small thermostable protein that is easily
over-expressed in bacterial systems. Thioredoxin fusion systems are
useful in avoiding the formation of inclusion bodies during
heterologous gene expression. This has been particularly useful in
the expression of mammalian cytokines.
[0103] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises Calmodulin Binding Protein (CBP) (SEQ ID NO:
12). This tag is derived from the C-terminus of skeletal muscle
myosin light chain kinase. This small tag is recognized by
calmodulin and forms the base of the technology. The tag is
translated efficiently and allows for the expression and recovery
of N-terminal chimeric genes.
[0104] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the c-myc epitope sequence
Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 13). This
C-terminal portion of the myc oncogene, which is part of the p53
signaling pathway, has been used as a detection tag for expression
of recombinant proteins in mammalian cells.
[0105] In another embodiment of this invention, a spacer (Sp.sub.1
or Sp.sub.2) comprises the HA epitope sequence
Tyr-Pro-Tyr-Asp-Val-Tyr-Ala (SEQ ID NO: 14). This detection tag has
been utilized for the expression of recombinant proteins in
mammalian cells.
[0106] In another embodiment of this invention, the spacer
(Sp.sub.1 or Sp.sub.2) comprises a polypeptide possessing an amino
acid sequence having at least 70% homology to any one of the amino
acid sequences disclosed in SEQ ID NOS:1-14, and retains the same
binding characteristics as said amino acid sequence.
[0107] DNA sequences encoding the aforementioned proteins which may
be employed as spacers (Sp.sub.1 or Sp.sub.2) are commercially
available (e.g., malE gene sequences encoding the MBP are available
from New England Biolabs (pMAL-c2 and pMAL-p2); Schistosoma
japonicum glutathione-S-transferase (GST) gene sequences are
available from Pharmacia Biotech (the pGEX series which have
GenBank Accession Nos.: U13849 to U13858); .beta.-galactosidase
(the lacZ gene product) gene sequences are available from Pharmacia
Biotech (pCH110 and pMC1871; GenBank Accession Nos: U13845 and
L08936, respectively); sequences encoding the IgG binding domains
of Protein A are available from Pharmacia Biotech (pRIT2T; GenBank
Accession No. U13864)).
[0108] When any of the above listed proteins (including the
hinge/Fc domains of human IgG.sub.1) are used as spacers, it is not
required that the entire protein be used as a spacer. Portions of
these proteins may be used as the spacer provided the portion
selected is sufficient to permit interaction of a fusion protein
containing the portion of the protein used as the spacer with the
desired affinity resin.
[0109] Expression and Purification
[0110] The polypeptides, proteins and protein fragments of the
present invention are generally prepared and expressed as a fusion
protein using conventional recombinant DNA technology. The fusion
protein is thus produced by host cells transformed with the genetic
information encoding the fusion protein. The host cells may secrete
the fusion protein into the culture media or store it in the cells
whereby the cells must be collected and disrupted in order to
extract the product. As hosts, E. coli, yeast, insect cells,
mammalian cells and plants are suitable. Of these two, E. coli will
typically be the more preferred host for most applications. In one
embodiment, the recombinant polypeptides, proteins and protein
fragments are produced in a soluble form or secreted from the
host.
[0111] In general, a chimeric gene is inserted into an expression
vector which allows for the expression of the desired fusion
protein in a suitable transformed host. The expression vector
provides the inserted chimeric gene with the necessary regulatory
sequences to control expression in the suitable transformed
host.
[0112] There are six elements of control expression sequence for
proteins which are to be secreted from a host into the medium,
while five of these elements apply to fusion proteins expressed
intracellularly. These elements in the order they appear in the
gene are: a) the promoter region; b) the 5' untranslated region; c)
signal sequence; d) the chimeric coding sequence; e) the 3'
untranslated region; f) the transcription termination site. Fusion
proteins which are not secreted do not contain c), the signal
sequence.
[0113] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression,
operably linked to the nucleic acid sequence to be expressed. It
will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. The expression vectors of the invention can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein.
[0114] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
For stable transfection of mammalian cells, it is known that,
depending upon the expression vector and transfection technique
used, only a small fraction of cells may integrate the foreign DNA
into their genome. In order to identify and select these
integrants, a gene that encodes a selectable marker (e.g., for
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin, and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding the metal ion-affinity peptide containing fusion
protein or can be introduced on a separate vector. Cells stably
transfected with the introduced nucleic acid can be identified by
drug selection (e.g., cells that have incorporated the selectable
marker gene will survive, while the other cells die). Methods and
materials for preparing recombinant vectors, transforming host
cells using replicating vectors, and expressing biologically active
foreign polypeptides and proteins are generally well known.
[0115] The expressed recombinant polypeptides, proteins and protein
fragments may be separated from other material present in the
secretion media or extraction solution, or from other liquid
mixtures, through immobilized metal affinity chromatography
("IMAC"). For example, the culture media containing the secreted
recombinant polypeptides, proteins and protein fragments or the
cell extracts containing the recombinant polypeptides, proteins and
protein fragments may be passed through a column that contains a
resin comprising an immobilized metal ion. In IMAC, metal ions are
immobilized onto to a solid support, and used to capture proteins
comprising a metal chelating peptide. The metal chelating peptide
may occur naturally in the protein, or the protein may be a
recombinant protein with an affinity tag comprising a metal
chelating peptide. Exemplary metal ions include aluminum, cadmium,
calcium, cobalt, copper, gallium, iron, nickel, ytterbium and zinc.
In one embodiment, the metal ion is preferably nickel, copper,
cobalt, or zinc. In another embodiment, the metal ion is nickel.
Advantageously, the components of the solution other than
recombinant polypeptide, protein or protein fragment freely pass
through the column. The immobilized metal, however, chelates or
binds the recombinant polypeptides, proteins and protein fragments,
thereby separating it from the remaining contents of the liquid
mixture in which it was originally contained.
[0116] Resins useful for producing immobilized metal ion affinity
chromatography (IMAC) columns are available commercially. Examples
of resins derivatized with iminodiacetic acid (IDA) are Chelating
Sepharose 6B (Pharmacia), Immobilized Iminodiacetic Acid (Pierce),
and Iminodiacetic Acid Agarose (Sigma-Aldrich). In addition, Porath
has immobilized tris(carboxymethyl)ethylenediamine (TED) on
Sepharose 6B and used it to fractionate serum proteins. Porath, J.
and Olin, B., Biochemistry, 22:1621-1630, 1983. Other reports
suggest that trisacryl GF2000 and silica can be derivatized with
IDA, TED, or aspartic acid, and the resulting materials used in
producing IMAC substances.
[0117] In one embodiment, the capture ligand is a metal chelate as
described in WO 01/81365. More specifically, in this embodiment the
capture ligand is a metal chelate derived from metal chelating
composition (1):
##STR00001##
wherein [0118] Q is a carrier; [0119] S.sup.1 is a spacer; [0120] L
is -A-T-CH(X)-- or --C(.dbd.O)--; [0121] A is an ether, thioether,
selenoether, or amide linkage; [0122] T is a bond or substituted or
unsubstituted alkyl or alkenyl; [0123] X is
--(CH.sub.2).sub.kCH.sub.3,
--(CH.sub.2).sub.kCOOH,--(CH.sub.2).sub.kSO.sub.3H,
--(CH.sub.2).sub.kPO.sub.3H.sub.2, --(CH.sub.2).sub.kN(J).sub.2, or
--(CH.sub.2).sub.kP(J).sub.2, preferably --(CH.sub.2).sub.kCOOH or
--(CH.sub.2).sub.kSO.sub.3H; [0124] k is an integer from 0 to 2;
[0125] J is hydrocarbyl or substituted hydrocarbyl; [0126] Y is
--COOH, --H, --SO.sub.3H, --PO.sub.3H.sub.2, --N(J).sub.2, or
--P(J).sub.2, preferably, --COOH; [0127] Z is --COOH, --H,
--SO.sub.3H, --PO.sub.3H.sub.2, --N(J).sub.2, or --P(J).sub.2,
preferably, --COOH; and [0128] i is an integer from 0 to 4,
preferably 1 or 2.
[0129] In general, the carrier, Q, may comprise any solid or
soluble material or compound capable of being derivatized for
coupling. Solid (or insoluble) carriers may be selected from a
group including agarose, cellulose, methacrylate co-polymers,
polystyrene, polypropylene, paper, polyamide, polyacrylonitrile,
polyvinylidene, polysulfone, nitrocellulose, polyester,
polyethylene, silica, glass, latex, plastic, gold, iron oxide and
polyacrylamide, but may be any insoluble or solid compound able to
be derivatized to allow coupling of the remainder of the
composition to the carrier, Q. Soluble carriers include proteins,
nucleic acids including DNA, RNA, and oligonucleotides, lipids,
liposomes, synthetic soluble polymers, proteins, polyamino acids,
albumin, antibodies, enzymes, streptavidin, peptides, hormones,
chromogenic dyes, fluorescent dyes, flurochromes or any other
detection molecule, drugs, small organic compounds, polysaccharides
and any other soluble compound able to be derivatized for coupling
the remainder of the composition to the carrier, Q. In one
embodiment, the carrier, Q, is the container of the present
invention. In another embodiment, the carrier, Q, is a body
provided within the container of the present invention.
[0130] The spacer, S.sup.1, which flanks the carrier comprises a
chain of atoms which may be saturated or unsaturated, substituted
or unsubstituted, linear or cyclic, or straight or branched.
Typically, the chain of atoms defining the spacer, S.sup.1, will
consist of no more than about 25 atoms; stated another way, the
backbone of the spacer will consist of no more than about 25 atoms.
More preferably, the chain of atoms defining the spacer, S.sup.1,
will consist of no more than about 15 atoms, and still more
preferably no more than about 12 atoms. The chain of atoms defining
the spacer, S.sup.1, will typically be selected from the group
consisting of carbon, oxygen, nitrogen, sulfur, selenium, silicon
and phosphorous and preferably from the group consisting of carbon,
oxygen, nitrogen, sulfur and selenium. In addition, the chain atoms
may be substituted or unsubstituted with atoms other than hydrogen
such as hydroxy, keto (.dbd.O), or acyl such as acetyl. Thus, the
chain may optionally include one or more ether, thioether,
selenoether, amide, or amine linkages between hydrocarbyl or
substituted hydrocarbyl regions. Exemplary spacers, S.sup.1,
include methylene, alkyleneoxy (--(CH.sub.2).sub.aO--),
alkylenethioether (--(CH.sub.2).sub.aS--), alkyleneselenoether
(--(CH.sub.2).sub.aSe--), alkyleneamide
(--(CH.sub.2).sub.aNR.sup.1(C.dbd.O)--), alkylenecarbonyl
(--(CH.sub.2).sub.aCO)--, and combinations thereof wherein a is
generally from 1 to about 20 and R.sup.1 is hydrogen or
hydrocarbyl, preferably alkyl. In one embodiment, the spacer,
S.sup.1, is a hydrophilic, neutral structure and does not contain
any amine linkages or substituents or other linkages or
substituents which could become electrically charged during the
purification of a polypeptide.
[0131] As noted above, the linker, L, may be -A-T-CH(X)-- or
--C(.dbd.O)--. When L is -A-T-CH(X)--, the chelating composition
corresponds to the formula:
##STR00002##
wherein Q, S.sup.1, A, T, X, Y, and Z are as previously defined. In
this embodiment, the ether (--O--), thioether (--S--), selenoether
(--Se--) or amide ((--NR.sup.1(C.dbd.O)--) or
(--(C.dbd.O)NR.sup.1--) wherein R.sup.1 is hydrogen or hydrocarbyl)
linkage is separated from the chelating portion of the molecule by
a substituted or unsubstituted alkyl or alkenyl region. If other
than a bond, T is preferably substituted or unsubstituted C.sub.1
to C.sub.6 alkyl or substituted or unsubstituted C.sub.2 to C.sub.6
alkenyl. More preferably, A is --S--, T is --(CH.sub.2).sub.n--,
and n is an integer from 0 to 6, typically 0 to 4, and more
typically 0, 1 or 2.
[0132] When L is --C(.dbd.O)--, the chelating composition
corresponds to the formula:
##STR00003##
wherein Q, S.sup.1, i, Y, and Z are as previously defined.
[0133] In one embodiment, the sequence --S.sup.1-L-, in
combination, is a chain of no more than about 35 atoms selected
from the group consisting of carbon, oxygen, sulfur, selenium,
nitrogen, silicon and phosphorous, more preferably only carbon,
oxygen sulfur and nitrogen, and still more preferably only carbon,
oxygen and sulfur. To reduce the prospects for non-specific
binding, nitrogen, when present, is preferably in the form of an
amide moiety. In addition, if the carbon chain atoms are
substituted with anything other than hydrogen, they are preferably
substituted with hydroxy or keto. In one embodiment, L comprises a
portion (sometimes referred to as a fragment or residue) derived
from an amino acid such as cystine, homocystine, cysteine,
homocysteine, aspartic acid, cysteic acid or an ester thereof such
as the methyl or ethyl ester thereof.
[0134] Exemplary chelating compositions corresponding to formula 1
include the following:
##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008##
wherein Q is a carrier and Ac is acetyl.
[0135] In another embodiment, the capture ligand is a metal chelate
of the type described in U.S. Pat. No. 5,047,513. More
specifically, in this embodiment the capture ligand is a metal
chelate derived from nitrilotriacetic acid derivatives of the
formula
##STR00009##
wherein S.sup.2 is --O--CH.sub.2--CH(OH)--CH.sub.2 or --O--CO-- and
x is 2, 3 or 4. In this embodiment, the nitrilotriacetic acid
derivative is immobilized on any of the previously described
carriers, Q.
[0136] In these embodiments in which the capture ligand is a metal
chelate as described in WO 01/81365 or U.S. Pat. No. 5,047,513, the
metal chelate may contain any of the metal ions previously
described in connection with IMAC. In one embodiment, the metal
chelate comprises a metal ion selected from among nickel
(Ni.sup.2+), zinc (Zn.sup.2+), copper (Cu.sup.2+), iron
(Fe.sup.3+), cobalt (Co.sup.2+), calcium (Ca.sup.2+), aluminum
(Al.sup.3+), magnesium (Mg.sup.2+), and manganese (Mn.sup.2+). In
another embodiment, the metal chelate comprises nickel
(Ni.sup.2+).
[0137] Another common purification technique that can be used in
the context of the present invention is the use of an immunogenic
capture system where the recombinant polypeptide, protein or
protein fragment comprises an antigenic domain in a spacer region
(Sp.sub.1 or Sp.sub.2). Any of the previously described antigenic
systems comprising the spacer may be used for this purpose. In such
systems, an epitope tag on a protein or peptide allows the protein
to which it is attached to be purified based upon the affinity of
the epitope tag for a corresponding ligand (e.g., antibody)
immobilized on a support. One example of such a tag is the sequence
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 15), or DYKDDDDK (SEQ
ID NO: 15); antibodies having specificity for this sequence are
sold by Sigma-Aldrich (St. Louis, Mo.) under the FLAG.RTM.
trademark. Another example of such a tag is the sequence
Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 16), or DLYDDDDK (SEQ
ID NO: 16); antibodies having specificity for this sequence are
sold by Invitrogen (Carlsbad, Calif.). Another example of such a
tag is the 3.times. FLAG.RTM. sequence
Met-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr-Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-A-
sp-Asp-Asp-Asp-Lys (SEQ ID NO: 17); antibodies having specificity
for this sequence are sold by Sigma-Aldrich (St. Louis, Mo.). Thus,
in one embodiment, the carrier comprises immobilized antibodies
which have specificity for the DYKDDDDK (SEQ ID NO: 15) epitope; in
another embodiment, the carrier comprises immobilized antibodies
which have specificity for the DLYDDDDK (SEQ ID NO: 16) epitope. In
another embodiment, the carrier comprises immobilized antibodies
which have specificity for SEQ ID NO: 17. For example, in one
embodiment, the ANTI-FLAG.RTM. M1, M2, or M5 antibody is
immobilized on the interior surface of a column, or a portion
thereof, and/or a bead or other support within a column.
[0138] After the recombinant polypeptides, proteins and protein
fragments are separated from other components of the liquid
mixture, the conditions in the column may be changed to release the
bound material. For example, the bound molecules may be eluted by
pH change, imidazole, or competition with another linker peptide
from the column.
[0139] Alternatively, the target polypeptide, protein or protein
fragment portion of the bound recombinant polypeptide, protein or
protein fragment may be selectively released from immobilized
metal. For example, if there is a cleavage site between the target
polypeptide, protein or protein fragment and the metal ion-affinity
peptide, and if the bound recombinant polypeptide, protein or
protein fragment is treated with the appropriate enzyme, the target
polypeptide, protein or protein fragment may be selectively
released while the metal ion-affinity polypeptide fragment remains
bound to the immobilized metal. For this purpose, the cleavage is
preferably an enzymatically cleavable linker peptide having the
ability to undergo site-specific proteolysis. Suitable cleaving
enzymes in accordance with this invention are activated factor X
(factor Xa), DPP I, DPP II, DPP IV, carboxylpeptidase A, collagen,
enterokinase, human renin, thrombin, trypsin, ubtilisn and V5.
[0140] It is to be appreciated that some polypeptide or protein
molecules will possess the desired enzymatic or biological activity
with the metal chelate peptide still attached either at the
C-terminal end or at the N-terminal end or both. In those cases the
purification of the chimeric protein will be accomplished without
subjecting the protein to site-specific proteolysis.
[0141] The present invention may be used to purify any prokaryotic
or eukaryotic protein that can be expressed as the product of
recombinant DNA technology in a transformed host cell. These
recombinant protein products include hormones, receptors, enzymes,
storage proteins, blood proteins, mutant proteins produced by
protein engineering techniques, or synthetic proteins. The
purification process of the present invention can be used batchwise
or in continuously run columns.
[0142] It is to be understood that the present invention has been
described in detail by way of illustration and example in order to
acquaint others skilled in the art with the invention, its
principles, and its practical application. Further, the specific
embodiments of the present invention as set forth are not intended
to be exhaustive or to limit the invention, and that many
alternatives, modifications, and variations will be apparent to
those skilled in the art in light of the foregoing examples and
detailed description. Accordingly, this invention is intended to
embrace all such alternatives, modifications, and variations that
fall within the spirit and scope of the following claims. While
some of the examples and descriptions above include some
conclusions about the way the invention may function, the inventors
do not intend to be bound by those conclusions and functions, but
put them forth only as possible explanations in light of current
understanding.
Abbreviations and Definitions
[0143] To facilitate understanding of the invention, a number of
terms are defined below. Definitions of certain terms are included
here. Any term not defined is understood to have the normal meaning
used by scientists contemporaneous with the submission of this
application.
[0144] The term "expression vector" as used herein refers to
nucleic acid sequences containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
include a promoter, a ribosome binding site, an initiation codon, a
stop codon, optionally an operator sequence and possibly other
regulatory sequences. Eukaryotic cells utilize promoters, a Kozak
sequence and often enhancers and polyadenlyation signals.
Prokaryotic cells also utilize a Shine-Dalgarno Ribosome binding
site. The present invention includes vectors or plasmids which can
be used as vehicles to transform any viable host cell with the
recombinant DNA expression vector.
[0145] "Operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner that allows for expression of the nucleotide sequence (e.g.,
in an in vitro transcription/translation system or in a host cell
when the vector is introduced into the host cell).
[0146] The term "regulatory sequence" is intended to include
promoters, enhancers, and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence in many
types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences).
[0147] The terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in laboratory manuals.
[0148] The term "hydrophilic" when used in reference to amino acids
refers to those amino acids which have polar and/or charged side
chains. Hydrophilic amino acids include lysine, arginine,
histidine, aspartate (i.e., aspartic acid), glutamate (i.e.,
glutamic acid), serine, threonine, cysteine, tyrosine, asparagine
and glutamine.
[0149] The term "hydrophobic" when used in reference to amino acids
refers to those amino acids which have nonpolar side chains.
Hydrophobic amino acids include valine, leucine, isoleucine,
cysteine and methionine. Three hydrophobic amino acids have
aromatic side chains. Accordingly, the term "aromatic" when used in
reference to amino acids refers to the three aromatic hydrophobic
amino acids phenylalanine, tyrosine and tryptophan.
[0150] The term "fusion protein" refers to polypeptides and
proteins which consist of a metal ion-affinity linker peptide and a
protein or polypeptide operably linked directly or indirectly to
the metal ion-affinity peptide. The metal ion-affinity linker
peptide may be located at the amino-terminal portion of the fusion
protein or at the carboxy-terminal protein thus forming an
"amino-terminal fusion protein" or a "carboxy-terminal fusion
protein," respectively.
[0151] The terms "metal ion-affinity peptide", "metal binding
peptide" and "linker peptide" are used interchangeably to refer to
an amino acid sequence which displays an affinity to metal ions.
The minimum length of the immobilized metal ion-affinity peptide
according to the present invention is seven amino acids including
four alternating histidines. The most preferred length is seven
amino acids including four alternating histidines.
[0152] The term "enzyme" referred to herein in the context of a
cleavage enzyme means a polypeptide or protein which recognizes a
specific amino acid sequence in a polypeptide and cleaves the
polypeptide at the scissile bond. In one embodiment of the present
invention, enterokinase is the enzyme which is used to free the
fusion protein from the immobilized metal ion column. In further
embodiments, carboxylpeptidase A, DPP I, DPP II, DPP IV, factor Xa,
human renin, TEV, thrombin or VIII protease is the enzyme.
[0153] The terms "cleavage site" used herein refers to an amino
acid sequence which is recognized and cleaved by an enzyme or
chemical means at the scissile bond.
[0154] The term "scissile bond" referred to herein is the juncture
where cleavage occurs; for example the scissile bond recognized by
enterokinase may be the bond following the sequence (Asp.sub.4)-Lys
in the spacer peptide or affinity peptide.
[0155] By the term "immobilized metal ion-affinity peptide" as used
herein is meant an amino acid sequence that chelates immobilized
divalent metal ions of metals selected from the group consisting of
aluminum, cadmium, calcium, cobalt, copper, gallium, iron, nickel,
ytterbium and zinc.
[0156] The term "capture ligand" means any ligand or receptor that
can be immobilized or supported on a container or support and used
to isolate a cellular component from cellular debris. Some
non-limiting examples of capture ligands that may be used in
connection with the present invention include: biotin,
streptavidin, various metal chelate ions, antibodies, various
charged particles such as those for use in ion exchange
chromatography, various affinity chromatography supports, and
various hydrophobic groups for use in hydrophobic
chromatography.
[0157] For all the nucleotide and amino acid sequences disclosed
herein, it is understood that equivalent nucleotides and amino
acids can be substituted into the sequences without affecting the
function of the sequences. Such substitutions are within the
ability of a person of ordinary skill in the art.
[0158] The procedures disclosed herein which involve the molecular
manipulation of nucleic acids are known to those skilled in the
art.
EXAMPLES
Example 1
Construction and Screening of a Metal Ion-Affinity Peptide
Library
[0159] A pseudo-random glutathione-S-transferase C-terminal peptide
library was constructed with the amino acid sequence of
His-X-His-X-His-X-His where X is any amino acid except Gln, His and
Pro. The library vector was constructed from the bacterial
expression vector pGEX-2T. The library was constructed by annealing
a pair of complimentary oligonucleotides together. Oligonucleotides
were constructed as follows: 5'GATCCCATDNDCATDNDCATDNDCATTAAC3'
(SEQ ID NO: 18) and 5'AATTGTTAATGHNHATGHNHATGHNHATGG3' (SEQ ID NO:
19) where D is nucleotides A, G, or T, H is nucleotides A, C, or T
and N is nucleotides A, C, T, or G. The 5' end was phosphorylated
with T.sub.4 polynucleotide kinase and the oligonucleotides were
annealed together to generate a cassette. The cassette was ligated
into pGEX-2T, which had been digested with EcoRI and BamHI
restriction endonucleases. Ligated vector was transformed into E.
coli DH5-.alpha. using standard protocols. Transformants were
plated on LB/ampicillin plates (100 mg/L) and incubated overnight
at 37.degree. C.
[0160] 900 colonies were picked and placed on 9 master plates. Each
master plate contained 100 colonies each and were grown overnight
at 37.degree. C. A piece of nitrocellulose was placed onto each of
the master plates. This piece of nitrocellulose was then removed
and the transferred colonies were placed onto a LB/ampicillin plate
containing 1 mM isopropyl .beta.-D-galactopyranoside (IPTG) to
induce the expression of the GST fusion peptides. The cells were
allowed to grow for an additional 4 hours at 37.degree. C. The
nitrocellulose filter was removed from the plate and placed
sequentially on blotting paper containing the following solutions
to lyse the cells in situ: [0161] (a) 10% SDS for 10 minutes,
[0162] (b) 1.5 M sodium chloride, 0.5 M sodium hydroxide for 5
minutes [0163] (c) 1.5 M sodium chloride, 0.5 M Tris-HCl pH 7.4 for
5 minutes [0164] (d) 1.5 M sodium chloride, 0.5 M Tris-HCl pH 7.4
for 5 minutes [0165] (e) 2.times.SSC for 15 minutes.
[0166] The filters were dried at ambient temperature followed by an
incubation in Tris-buffered saline (TBS) containing 3% non-fat dry
milk for 1 hour at room temperature. Filters were then washed
3.times. for 5 minutes with TBS containing 0.05% Tween-20 (TBS-T).
To detect clones that were capable of binding to a metal ion, the
filters were incubated with nickel NTA horseradish peroxidase (HRP)
at a concentration of 1 mg/ml in TBS-T for 1 hour. The filters were
then washed with TBS-T 3.times. for 5 minutes and incubated with
3-3'-5-5'-Tetramethylbenzidine (TMB) to detect the horseradish
peroxidase. The reaction was stopped by placing the filters in
water. 250 colonies, which were detected above, were picked from
the master plate and placed into 1 ml of LB/ampicillin and grown
overnight in a 96 deep well plate at 37.degree. C. at 250 rpm on an
orbital shaker. 10 .mu.l of the overnight cultures were transferred
to a fresh aliquot of LB/ampicillin (1 ml) in a 96 deep well plate
and grown for an additional 3 hours. The culture was then induced
by adding IPTG (final concentration of 1 mM) and the culture was
allowed to grow for an additional 3 hours prior to harvesting by
centrifugation. The media was decanted and the cells were frozen
overnight at -20.degree. C. in the collection plate. Cells were
lysed with 0.6 ml of CelLytic-B (Sigma-Aldrich product no. B3553)
and incubated for 15 minutes at room temperature. The cell debris
was removed by centrifugation at 3,000.times.g for 15 minutes. Two
experiments were done in parallel, one on a His-Select High
Sensitivity (HS) nickel coated plate, and the second on HIS-Select
High Capacity (HC) nickel coated plate. 0.1 ml of cell extracts of
each clone were placed in a HS microwell plate in the presence of
imidazole at a final concentration of 5 mM. This is the selective
condition used for screening the different metal ion-affinity
clones. HS plates were incubated for 4 hours at room temperature.
Plates were then washed 3.times. with phosphate-buffered saline
(PBS) containing 0.05% Tween 20 (PBS-T). The HS plates were then
incubated with anti-GST at 1:1,000 dilution in PBS-BSA buffer (0.2
ml/well) for 1 hour at room temperature. HS plates were washed
3.times. with PBS-T. The HS plates were then incubated with
anti-mouse HRP conjugate at 1:10,000 dilution in PBS-BSA buffer for
1 hour at room temperature. Plates were washed 3.times. with PBS-T.
The plate was then developed with
2,2'azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABST
substrate. Color development was stopped by the addition of sodium
azide to a final concentration of 2 mM. Absorbance of the plates
was read at 405 nm using a Wallace 1420 plate reader. The HC plates
were used to further analyze potential clones. To further
characterize the clones, 0.2 ml of cell extracts were applied to
the HC plates and the plates were incubated at ambient temperature
for 1 hour. The plates were washed with PBS as described above.
Twenty-one clones that produced the highest response on the HS
plates were eluted from the corresponding HC plate. The selected
cloned proteins were eluted from the HC plates by incubating at
37.degree. C. for 15 minutes in 50 mM sodium phosphate, 0.3 M
sodium chloride and 0.2 M imidazole buffer. Eluted proteins were
then moved to clean tubes and analyzed by SDS-PAGE. All 21 clones
had the expected molecular weight and were sequence verified.
[0167] These 21 colonies were grown overnight in 1 ml LB/ampicillin
media at 37.degree. C. at 250 rpm. 100 .mu.l of the overnight
cultures were transferred to 50 ml of fresh LB/ampicillin media and
the cultures grown for an additional 3 hours at 37.degree. C. The
cultures were induced with IPTG (final concentration of 1 mM) and
the cultures grown for an additional 3 hours prior to harvesting by
centrifugation.
Example 2
Construction of a N-Terminal Metal Ion-Affinity Fusion Protein
[0168] Two metal ion-affinity tags were introduced to the
N-terminal of bacterial alkaline phosphatase (BAP). The constructs
were constructed from the BAP expression vector pFLAG-CTS-BAP.
Construction was done by annealing two pair of complimentary
oligonucleotides together. The following oligonucleotides were
constructed: 5'TATGCATAATCATCGACATGAACATA3'(SEQ ID NO: 20),
5'AGCTTATGTTTATGTCGATGATTATGCA3' (SEQ ID NO: 21),
5'TATGCATAAACATAGACATGGGCATA3' (SEQ ID NO: 22) and
5'AGCTTGATGCCCATGTCTATGTTTATGCA3' (SEQ ID NO: 23). The
oligonucleotides were annealed together to generate a cassette. The
cassette was ligated into pFLAG-CTS-BAP, which had been digested
with NdeI and HindIII restriction endonucleases. Ligated vector was
transformed into E. coli DH5-a using standard protocols and plated
on LB/ampicillin.
Example 3
Expression of an N-Terminal Metal Ion-Affinity Fusion Protein
[0169] MAT-BAP fusion peptide cultures were grown overnight in 1 ml
LB/ampicillin at 37.degree. C. 500 .mu.l of overnight cultures were
transferred to 500 ml of fresh TB media containing ampicillin (100
mg/L). The cultures were grown for three hours at 37.degree. C. at
250 rpm. Protein expression was induced by the addition of IPTG
(final concentration of 1 mM). Cultures were then grown for an
additional three hours, harvested by centrifugation and stored at
-70.degree. C. until further use.
Example 4
Metal Ion-Affinity Fusion Protein Purification Protocol #1
[0170] Cells were resuspended in 2 ml of TE (50 mM Tris-HCl pH 8.0,
2 mM EDTA). Lysozyme (4 mg/ml in 2 ml of TE) was added to the
resuspended cells and the cells were lysed at ambient temperature
for 4 hours. The cell debris was removed by centrifugation at
27,000.times.g for 15 minutes. The supernatant was dialyzed
overnight against 50 mM Tris-HCl pH 8.0 to remove the EDTA. The
dialyzed supernatant was applied to a 1 ml column containing a
nickel bis-carboxy-methyl-cysteine resin (nickel resin). The column
was washed with 4 ml of 50 mM Tris-HCl pH 8.0 and then washed with
2 ml of 50 mM Tris-HCl pH 8.0, 10 mM imidazole. The column was then
eluted 50 mM Tris-HCl pH 8.0 250 mM imidazole. Samples were
analyzed for purity by SDS-PAGE.
Example 5
Metal Ion-Affinity Fusion protein Purification Protocol #2
[0171] Cells were resuspended with CelLytic B (Sigma-Aldrich
product no. B3553), and 10 mM imidazole. The cells were solubilized
by incubation for 15 minutes. The cell debris was removed by
centrifugation at 15,000.times.g for 5 minutes at room temperature.
A 0.5 ml column, containing nickel resin, was equilibrated with 10
column volumes (5 ml) of 50 mM sodium phosphate, pH 8, and 300 mM
sodium chloride (column buffer). The supernatant was loaded on the
column. The column was washed with 10 column volumes (5 ml) of 10
mM imidazole in column buffer. The column was eluted with 100 mM
imidazole in column buffer. The samples were analyzed for
specificity by SDS-PAGE.
Example 6
Metal Ion-Affinity Fusion Protein Purification Protocol #3: Use of
Chaotropic Agents
[0172] The cells were resuspended in 100 mM sodium phosphate, pH 8,
and 8 M urea (denaturant column buffer). The cells were solubilized
by sonication three times, 15 seconds each, with a probe sonicator.
Cell debris was removed by centrifugation at 15,000.times.g for 5
minutes at room temperature. A 0.5 ml column, containing nickel
resin, was equilibrated with 10 column volumes (5 ml) of the
denaturant column buffer. The supernatant was loaded on the column
and the column was washed with 10 column volumes (5 ml) of
denaturant column buffer. The column was sequentially eluted with
100 mM sodium phosphate, 8 M urea at pH 7.5, 7.0, 6.5, 6.0, 5.5,
5.0 and 4.5. The samples were analyzed for specificity by SDS-PAGE.
Sequence CWU 1
1
481211PRTSchistosoma japonicum 1Met Ala Cys Gly His Val Lys Leu Ile
Tyr Phe Asn Gly Arg Gly Arg1 5 10 15Ala Glu Pro Ile Arg Met Ile Leu
Val Ala Ala Gly Val Glu Phe Glu 20 25 30Asp Glu Arg Ile Glu Phe Gln
Asp Trp Pro Lys Ile Lys Pro Thr Ile 35 40 45Pro Gly Gly Arg Leu Pro
Ile Val Lys Ile Thr Asp Lys Arg Gly Asp 50 55 60Val Lys Thr Met Ser
Glu Ser Leu Ala Ile Ala Arg Phe Ile Ala Arg65 70 75 80Lys His Asn
Met Met Gly Asp Thr Asp Asp Glu Tyr Tyr Ile Ile Glu 85 90 95Lys Met
Ile Gly Gln Val Glu Asp Val Glu Ser Asp Tyr His Lys Thr 100 105
110Leu Ile Lys Pro Pro Glu Glu Lys Glu Lys Ile Ser Lys Glu Ile Leu
115 120 125Asn Gly Lys Val Pro Ile Leu Leu Gln Ala Ile Cys Glu Thr
Leu Lys 130 135 140Glu Ser Thr Gly Asn Leu Thr Val Gly Asp Lys Val
Thr Leu Ala Asp145 150 155 160Val Val Leu Ile Ala Ser Ile Asp His
Ile Thr Asp Leu Asp Lys Glu 165 170 175Phe Leu Thr Gly Lys Tyr Pro
Glu Ile His Lys His Arg Lys His Leu 180 185 190Leu Ala Thr Ser Pro
Lys Leu Ala Lys Tyr Leu Ser Glu Arg His Ala 195 200 205Thr Ala Phe
2102163PRTClostridium cellulovorans 2Ala Ala Thr Ser Ser Met Ser
Val Glu Phe Tyr Asn Ser Asn Lys Ser1 5 10 15Ala Gln Thr Asn Ser Ile
Thr Pro Ile Ile Lys Ile Thr Asn Thr Ser 20 25 30Asp Ser Asp Leu Asn
Leu Asn Asp Val Lys Val Arg Tyr Thr Tyr Tyr 35 40 45Thr Ser Asp Gly
Thr Gln Gly Gln Thr Phe Trp Cys Asp His Ala Gly 50 55 60Ala Leu Leu
Gly Asn Ser Tyr Val Asp Asn Thr Ser Lys Val Thr Ala65 70 75 80Asn
Phe Val Lys Glu Thr Ala Ser Pro Thr Ser Thr Tyr Asp Thr Tyr 85 90
95Val Glu Phe Gly Phe Ala Ser Gly Ala Ala Thr Leu Lys Lys Gly Gln
100 105 110Phe Ile Thr Ile Gln Gly Arg Ile Thr Lys Ser Asp Trp Ser
Asn Tyr 115 120 125Thr Gln Thr Asn Asp Tyr Ser Phe Asp Ala Ser Ser
Ser Thr Pro Val 130 135 140Val Asn Pro Lys Val Thr Gly Tyr Ile Gly
Gly Ala Lys Val Leu Gly145 150 155 160Thr Ala Pro3396PRTEscherichia
coli 3Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu
Thr1 5 10 15Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly
Leu Ala Glu 35 40 45Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys
Val Thr Val Glu 50 55 60His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln
Val Ala Ala Thr Gly65 70 75 80Asp Gly Pro Asp Ile Ile Phe Trp Ala
His Asp Arg Phe Gly Gly Tyr 85 90 95Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala145 150 155
160Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu225 230 235 240Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn305 310 315 320Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365Val Arg Thr Ala Val
Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp 370 375 380Glu Ala Leu
Lys Asp Ala Gln Thr Arg Ile Thr Lys385 390 3954524PRTStaphylococcus
aureus 4Met Lys Lys Lys Asn Ile Tyr Ser Ile Arg Lys Leu Gly Val Gly
Ile1 5 10 15Ala Ser Val Thr Leu Gly Thr Leu Leu Ile Ser Gly Gly Val
Thr Pro 20 25 30Ala Ala Asn Ala Ala Gln His Asp Glu Ala Gln Gln Asn
Ala Phe Tyr 35 40 45Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln
Arg Asn Gly Phe 50 55 60Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser
Ala Asn Val Leu Gly65 70 75 80Glu Ala Gln Lys Leu Asn Asp Ser Gln
Ala Pro Lys Ala Asp Ala Gln 85 90 95Gln Asn Asn Phe Asn Lys Asp Gln
Gln Ser Ala Phe Tyr Glu Ile Leu 100 105 110Asn Met Pro Asn Leu Asn
Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser 115 120 125Leu Lys Asp Asp
Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys 130 135 140Lys Leu
Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys145 150 155
160Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn
165 170 175Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp
Pro Ser 180 185 190Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys Leu
Asn Glu Ser Gln 195 200 205Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys
Glu Gln Gln Asn Ala Phe 210 215 220Tyr Glu Ile Leu His Leu Pro Asn
Leu Asn Glu Glu Gln Arg Asn Gly225 230 235 240Phe Ile Gln Ser Leu
Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 245 250 255Ala Glu Ala
Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn 260 265 270Lys
Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 275 280
285Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys
290 295 300Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys
Lys Leu305 310 315 320Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn
Asn Lys Pro Gly Lys 325 330 335Glu Asp Asn Asn Lys Pro Gly Lys Glu
Asp Asn Asn Lys Pro Gly Lys 340 345 350Glu Asp Asn Asn Lys Pro Gly
Lys Glu Asp Asn Asn Lys Pro Gly Lys 355 360 365Glu Asp Asn Asn Lys
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys 370 375 380Glu Asp Asn
Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys385 390 395
400Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys
405 410 415Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Gly Val
His Val 420 425 430Val Lys Pro Gly Asp Thr Val Asn Asp Ile Ala Lys
Ala Asn Gly Thr 435 440 445Thr Ala Asp Lys Ile Ala Ala Asp Asn Lys
Leu Ala Asp Lys Asn Met 450 455 460Ile Lys Pro Gly Gln Glu Leu Val
Val Asp Lys Lys Gln Pro Ala Asn465 470 475 480His Ala Asp Ala Asn
Lys Ala Gln Ala Leu Pro Glu Thr Gly Glu Glu 485 490 495Asn Pro Phe
Ile Gly Thr Thr Val Phe Gly Gly Leu Ser Leu Ala Leu 500 505 510Gly
Ala Ala Leu Leu Ala Gly Arg Arg Arg Glu Leu 515
5205448PRTStreptococcus pyogenes 5Met Glu Lys Glu Lys Lys Val Lys
Tyr Phe Leu Arg Lys Ser Ala Phe1 5 10 15Gly Leu Ala Ser Val Ser Ala
Ala Phe Leu Val Gly Ser Thr Val Phe 20 25 30Ala Val Asp Ser Pro Ile
Glu Asp Thr Pro Ile Ile Arg Asn Gly Gly 35 40 45Glu Leu Thr Asn Leu
Leu Gly Asn Ser Glu Thr Thr Leu Ala Leu Arg 50 55 60Asn Glu Glu Ser
Ala Thr Ala Asp Leu Thr Ala Ala Ala Val Ala Asp65 70 75 80Thr Val
Ala Ala Ala Ala Ala Glu Asn Ala Gly Ala Ala Ala Trp Glu 85 90 95Ala
Ala Ala Ala Ala Asp Ala Leu Ala Lys Ala Lys Ala Asp Ala Leu 100 105
110Lys Glu Phe Asn Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile
115 120 125Asn Asn Ala Lys Thr Val Glu Gly Ile Lys Asp Leu Gln Ala
Gln Val 130 135 140Val Glu Ser Ala Lys Lys Ala Arg Ile Ser Glu Ala
Thr Asp Gly Leu145 150 155 160Ser Asp Phe Leu Lys Ser Gln Thr Pro
Ala Glu Asp Thr Val Lys Ser 165 170 175Ile Glu Leu Ala Glu Ala Lys
Val Leu Ala Asn Arg Glu Leu Asp Lys 180 185 190Tyr Gly Val Ser Asp
Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr 195 200 205Val Glu Gly
Val Lys Glu Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro 210 215 220Lys
Thr Asp Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly225 230
235 240Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val
Phe 245 250 255Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp
Thr Tyr Asp 260 265 270Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys
Pro Glu Val Ile Asp 275 280 285Ala Ser Glu Leu Thr Pro Ala Val Thr
Thr Tyr Lys Leu Val Ile Asn 290 295 300Gly Lys Thr Leu Lys Gly Glu
Thr Thr Thr Lys Ala Val Asp Ala Glu305 310 315 320Thr Ala Glu Lys
Ala Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp 325 330 335Gly Val
Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu 340 345
350Met Val Thr Glu Val Pro Gly Asp Ala Pro Thr Glu Pro Glu Lys Pro
355 360 365Glu Ala Ser Ile Pro Leu Val Pro Leu Thr Pro Ala Thr Pro
Ile Ala 370 375 380Lys Asp Asp Ala Lys Lys Asp Asp Thr Lys Lys Glu
Asp Ala Lys Lys385 390 395 400Pro Glu Ala Lys Lys Asp Asp Ala Lys
Lys Ala Glu Thr Leu Pro Thr 405 410 415Thr Gly Glu Gly Ser Asn Pro
Phe Phe Thr Ala Ala Ala Leu Ala Val 420 425 430Met Ala Gly Ala Gly
Ala Leu Ala Val Ala Ser Lys Arg Lys Glu Asp 435 440 4456192PRTHomo
sapiens 6Met Ala Pro Ser Leu Ser Ala Met Thr Pro Trp Thr Pro Gly
Pro Ser1 5 10 15Trp Ser Ser Val Tyr Met Thr Cys Val Trp Ser Val Gly
Ser Gly Ser 20 25 30Ala Cys Ala Val Ala Ser Ala Pro Met Pro Arg Pro
Val Trp Ser Leu 35 40 45Ala Ser Arg Leu Gly Thr Gly Asp His Gln Pro
Thr Ala Pro Cys Pro 50 55 60Ala Leu Pro Thr Ala Ala Met Ser Ser Ala
Ala Leu Leu Ala Arg Pro65 70 75 80Pro Ala Thr Gly Leu Arg Arg Arg
Pro Thr Ala Pro Gly Ala Pro Ala 85 90 95Trp Arg Ala Ala Cys Ala Ser
Gln Ala Ser Trp Pro Ala Ala Ala Pro 100 105 110Ala Cys Arg Pro Arg
Arg Val Ala Ala Pro Ser Arg Val Ser Ser Ser 115 120 125Leu Arg Ala
Arg Lys Cys Gly Arg Thr Ser Cys Ala Lys Gly Ala Ala 130 135 140Pro
Ala Thr Ala Pro Pro Ile Arg Ser Pro Ala Ala Thr Ser Arg Ala145 150
155 160Ala Arg Arg Val Ser Ala Ala Ala Ser Arg Thr Ala Ser Trp Ala
Ala 165 170 175Thr Pro Ile Ala Ser Gly Pro Ala Arg Gly Pro Gly Thr
His Thr Met 180 185 1907216PRTEscherichia coli 7Met Asn Phe Asn Lys
Ile Asp Leu Asp Asn Trp Lys Arg Lys Glu Ile1 5 10 15Phe Asn His Tyr
Leu Asn Gln Gln Thr Thr Phe Ser Ile Thr Thr Glu 20 25 30Ile Asp Ile
Ser Val Leu Tyr Arg Asn Ile Lys Gln Glu Gly Tyr Lys 35 40 45Phe Tyr
Pro Ala Phe Ile Phe Leu Val Thr Arg Val Ile Asn Ser Asn 50 55 60Thr
Ala Phe Arg Thr Gly Tyr Asn Ser Asp Gly Glu Leu Gly Tyr Trp65 70 75
80Asp Lys Leu Glu Pro Leu Tyr Thr Ile Phe Asp Gly Val Ser Lys Thr
85 90 95Phe Ser Gly Ile Trp Thr Pro Val Lys Asn Asp Phe Lys Glu Phe
Tyr 100 105 110Asp Leu Tyr Leu Ser Asp Val Glu Lys Tyr Asn Gly Ser
Gly Lys Leu 115 120 125Phe Pro Lys Thr Pro Ile Pro Glu Asn Ala Phe
Ser Leu Ser Ile Ile 130 135 140Pro Trp Thr Ser Phe Thr Gly Phe Asn
Leu Asn Ile Asn Asn Asn Ser145 150 155 160Asn Tyr Leu Leu Pro Ile
Ile Thr Ala Gly Lys Phe Ile Asn Lys Gly 165 170 175Asn Ser Ile Tyr
Leu Pro Leu Ser Leu Gln Val His His Ser Val Cys 180 185 190Asp Gly
Tyr His Ala Gly Leu Phe Met Asn Ser Ile Gln Glu Leu Ser 195 200
205Asp Arg Pro Asn Asp Trp Leu Leu 210 2158160PRTStreptomyces
avidinii 8Met Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala
Glu Ala1 5 10 15Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr
Phe Ile Val 20 25 30Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr
Glu Ser Ala Val 35 40 45Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly
Arg Tyr Asp Ser Ala 50 55 60Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu
Gly Trp Thr Val Ala Trp65 70 75 80Lys Asn Asn Tyr Arg Asn Ala His
Ser Ala Thr Thr Trp Ser Gly Gln 85 90 95Tyr Val Gly Gly Ala Glu Ala
Arg Ile Asn Thr Gln Trp Leu Leu Thr 100 105 110Ser Gly Thr Thr Glu
Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His 115 120 125Asp Thr Phe
Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala 130 135 140Lys
Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln145 150
155 16091024PRTEscherichia coli 9Met Thr Met Ile Thr Asp Ser Leu
Ala Val Val Leu Gln Arg Arg Asp1 5 10 15Trp Glu Asn Pro Gly Val Thr
Gln Leu Asn Arg Leu Ala Ala His Pro 20 25 30Pro Phe Ala Ser Trp Arg
Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro 35 40 45Ser Gln Gln Leu Arg
Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe 50 55 60Pro Ala Pro Glu
Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro65 70 75 80Glu Ala
Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr 85 90 95Asp
Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro 100 105
110Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe
115 120 125Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile
Ile Phe 130 135
140Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp
Val145 150 155 160Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe
Asp Leu Ser Ala 165 170 175Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala
Val Met Val Leu Arg Trp 180 185 190Ser Asp Gly Ser Tyr Leu Glu Asp
Gln Asp Met Trp Arg Met Ser Gly 195 200 205Ile Phe Arg Asp Val Ser
Leu Leu His Lys Pro Thr Thr Gln Ile Ser 210 215 220Asp Phe His Val
Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala Val225 230 235 240Leu
Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg 245 250
255Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr
260 265 270Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr
Ala Asp 275 280 285Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys
Leu Trp Ser Ala 290 295 300Glu Ile Pro Asn Leu Tyr Arg Ala Val Val
Glu Leu His Thr Ala Asp305 310 315 320Gly Thr Leu Ile Glu Ala Glu
Ala Cys Asp Val Gly Phe Arg Glu Val 325 330 335Arg Ile Glu Asn Gly
Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile 340 345 350Arg Gly Val
Asn Arg His Glu His His Pro Leu His Gly Gln Val Met 355 360 365Asp
Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn 370 375
380Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp
Tyr385 390 395 400Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp
Glu Ala Asn Ile 405 410 415Glu Thr His Gly Met Val Pro Met Asn Arg
Leu Thr Asp Asp Pro Arg 420 425 430Trp Leu Pro Ala Met Ser Glu Arg
Val Thr Arg Met Val Gln Arg Asp 435 440 445Arg Asn His Pro Ser Val
Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly 450 455 460His Gly Ala Asn
His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp465 470 475 480Pro
Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala 485 490
495Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro
500 505 510Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser
Leu Pro 515 520 525Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala
His Ala Met Gly 530 535 540Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp
Gln Ala Phe Arg Gln Tyr545 550 555 560Pro Arg Leu Gln Gly Gly Phe
Val Trp Asp Trp Val Asp Gln Ser Leu 565 570 575Ile Lys Tyr Asp Glu
Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp 580 585 590Phe Gly Asp
Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val 595 600 605Phe
Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln 610 615
620Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val
Thr625 630 635 640Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu
Leu His Trp Met 645 650 655Val Ala Leu Asp Gly Lys Pro Leu Ala Ser
Gly Glu Val Pro Leu Asp 660 665 670Val Ala Pro Gln Gly Lys Gln Leu
Ile Glu Leu Pro Glu Leu Pro Gln 675 680 685Pro Glu Ser Ala Gly Gln
Leu Trp Leu Thr Val Arg Val Val Gln Pro 690 695 700Asn Ala Thr Ala
Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln705 710 715 720Trp
Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His 725 730
735Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu
740 745 750Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu
Ser Gln 755 760 765Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro
Leu Arg Asp Gln 770 775 780Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile
Gly Val Ser Glu Ala Thr785 790 795 800Arg Ile Asp Pro Asn Ala Trp
Val Glu Arg Trp Lys Ala Ala Gly His 805 810 815Tyr Gln Ala Glu Ala
Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala 820 825 830Asp Ala Val
Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys 835 840 845Thr
Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln 850 855
860Met Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His
Pro865 870 875 880Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val
Ala Glu Arg Val 885 890 895Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn
Tyr Pro Asp Arg Leu Thr 900 905 910Ala Ala Cys Phe Asp Arg Trp Asp
Leu Pro Leu Ser Asp Met Tyr Thr 915 920 925Pro Tyr Val Phe Pro Ser
Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu 930 935 940Leu Asn Tyr Gly
Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile945 950 955 960Ser
Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu 965 970
975Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met
980 985 990Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala
Glu Phe 995 1000 1005Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu
Val Trp Cys Gln 1010 1015 1020Lys10238PRTAequorea victoria 10Met
Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1 5 10
15Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu
20 25 30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
Cys 35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
Thr Phe 50 55 60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
Met Lys Gln65 70 75 80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
Tyr Val Gln Glu Arg 85 90 95Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140Tyr Asn Ser His
Asn Val Tyr Ile Met Ala Asp Lys Pro Lys Asn Gly145 150 155 160Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170
175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu
Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp
Glu Leu Tyr Lys225 230 23511109PRTEscherichia coli 11Met Ser Asp
Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp1 5 10 15Val Leu
Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30Cys
Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40
45Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu
Leu65 70 75 80Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly
Ala Leu Ser 85 90 95Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu
Ala 100 1051213PRTOryctolagus cuniculus 12Ile Ala Val Ser Ala Ala
Asn Arg Phe Lys Lys Ile Ser1 5 101310PRTArtificial
SequenceSYNTHESIZED 13Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu1 5
10147PRTArtificial SequenceSYNTHESIZED 14Tyr Pro Tyr Asp Val Tyr
Ala1 5158PRTArtificial SequenceSYNTHESIZED 15Asp Tyr Lys Asp Asp
Asp Asp Lys1 5168PRTArtificial SequenceSYNTHESIZED 16Asp Leu Tyr
Asp Asp Asp Asp Lys1 51723PRTArtificial SequenceSYNTHESIZED 17Met
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp1 5 10
15Tyr Lys Asp Asp Asp Asp Lys 201830DNAArtificial
SequenceSYNTHESIZED 18gatcccatdn dcatdndcat dndcattaac
301930DNAArtificial SequenceSYNTHESIZED 19aattgttaat ghnhatghnh
atghnhatgg 302026DNAArtificial SequenceSYNTHESIZED 20tatgcataat
catcgacatg aacata 262128DNAArtificial SequenceSYNTHESIZED
21agcttatgtt tatgtcgatg attatgca 282226DNAArtificial
SequenceSYNTHESIZED 22tatgcataaa catagacatg ggcata
262329DNAArtificial SequenceSYNTHESIZED 23agcttgatgc ccatgtctat
gtttatgca 29247PRTArtificial SequenceSYNTHESIZED 24His Xaa His Arg
His Xaa His1 5254PRTArtificial SequenceSYNTHESIZED 25Asp Asp Asp
Lys1265PRTArtificial SequenceSYNTHESIZED 26Asp Asp Asp Asp Lys1
5276PRTArtificial SequenceSYNTHESIZED 27Leu Val Pro Arg Gly Xaa1
5285PRTArtificial SequenceSYNTHESIZED 28Ile Glu Gly Arg Xaa1
5295PRTArtificial SequenceSYNTHESIZED 29Ile Asp Gly Arg Xaa1
5305PRTArtificial SequenceSYNTHESIZED 30Ala Glu Gly Arg Xaa1
5316PRTArtificial SequenceSYNTHESIZED 31Asp Tyr Lys Xaa Xaa Asp1
5328PRTArtificial SequenceSYNTHESIZED 32Asp Tyr Lys Xaa Xaa Asp Xaa
Lys1 5335PRTArtificial SequenceSYNTHESIZED 33Asp Xaa Tyr Xaa Xaa1
5348PRTArtificial SequenceSYNTHESIZED 34Asp Xaa Tyr Xaa Xaa Asp Xaa
Lys1 5357PRTArtificial SequenceSYNTHESIZED 35Xaa Tyr Lys Xaa Xaa
Asp Xaa1 5366PRTArtificial SequenceSYNTHESIZED 36Xaa Tyr Lys Xaa
Xaa Asp1 5378PRTArtificial SequenceSYNTHESIZED 37Xaa Tyr Lys Xaa
Xaa Asp Xaa Lys1 5385PRTArtificial SequenceSYNTHESIZED 38Xaa Xaa
Asp Xaa Lys1 53916PRTArtificial SequenceSYNTHESIZED 39Xaa Tyr Lys
Xaa Xaa Asp Xaa Xaa Xaa Tyr Lys Xaa Xaa Asp Xaa Lys1 5 10
154021PRTArtificial SequenceSYNTHESIZED 40Asp Tyr Lys Xaa Xaa Asp
Asp Tyr Lys Xaa Xaa Asp Xaa Asp Tyr Lys1 5 10 15Xaa Xaa Asp Xaa Lys
204112PRTArtificial SequenceSYNTHESIZED 41Asp Tyr Lys Xaa Xaa Asp
Asp Tyr Lys Xaa Xaa Asp1 5 104214PRTArtificial SequenceSYTHESIZED
42Asp Xaa Tyr Xaa Xaa Xaa Asp Xaa Tyr Xaa Xaa Asp Xaa Lys1 5
104310PRTArtificial SequenceSYNTHESIZED 43Asp Xaa Tyr Xaa Xaa Asp
Xaa Tyr Xaa Xaa1 5 104413PRTArtificial SequenceSYNTHESIZED 44Xaa
Asp Tyr Lys Xaa Xaa Asp Asp Tyr Lys Xaa Xaa Asp1 5
104522PRTArtificial SequenceSYNTHESIZED 45Xaa Asp Tyr Lys Xaa Xaa
Asp Asp Tyr Lys Xaa Xaa Asp Xaa Asp Tyr1 5 10 15Lys Xaa Xaa Asp Xaa
Lys 204623PRTArtificial SequenceSYNTHESIZED 46Met Asp Tyr Lys Asp
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp
Asp Asp Lys 20479PRTArtificial SequenceSYNTHESIZED 47Xaa Xaa Tyr
Lys Xaa Xaa Asp Xaa Lys1 5489PRTArtificial SequenceSYNTHESIZED
48Xaa Asp Xaa Tyr Xaa Xaa Asp Xaa Lys1 5
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