U.S. patent application number 10/672108 was filed with the patent office on 2004-02-26 for novel reagents for detection and purification of antibody fragments.
Invention is credited to Hellinga, Homme W., Sloan, David J..
Application Number | 20040038378 10/672108 |
Document ID | / |
Family ID | 23271812 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038378 |
Kind Code |
A1 |
Hellinga, Homme W. ; et
al. |
February 26, 2004 |
Novel reagents for detection and purification of antibody
fragments
Abstract
An isolated B1 domain polypeptide of bacterial Protein G which
binds a Fab fragment of an IgG but substantially does not bind a Fc
fragment of an IgG. Methods for the detection and purification of
IgG Fc antibody fragments and Fab antibody fragments using the
isolated GB1 domain polypeptides are also disclosed.
Inventors: |
Hellinga, Homme W.; (Durham,
NC) ; Sloan, David J.; (Durham, NC) |
Correspondence
Address: |
JENKINS & WILSON, PA
3100 TOWER BLVD
SUITE 1400
DURHAM
NC
27707
US
|
Family ID: |
23271812 |
Appl. No.: |
10/672108 |
Filed: |
September 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10672108 |
Sep 25, 2003 |
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09326342 |
Jun 4, 1999 |
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6663862 |
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Current U.S.
Class: |
435/252.3 |
Current CPC
Class: |
C07K 14/315 20130101;
C07K 2317/55 20130101; C07K 14/31 20130101; C07K 16/065 20130101;
C07K 2317/52 20130101 |
Class at
Publication: |
435/252.3 |
International
Class: |
C12N 001/20 |
Goverment Interests
[0001] This workwas supported by grant N00014-98-1-0110 from the
Office of Naval Research. Thus, the United States Government has
certain rights in the invention.
Claims
What is claimed is:
1. An isolated GB1 domain polypeptide which exhibits binding
activity for a Fab fragment of an IgG but exhibits substantially no
binding activity for a Fc fragment of an IgG.
2. The isolated GB1 domain polypeptide of claim 1, further
comprising a disassociation constant for a Fc fragment of an IgG of
greater than about 2 mM.
3. The isolated GB1 domain polypeptide of claim 1, further
comprising a disrupted "knobs-into-holes" binding site for a Fc
fragment of an IgG.
4. The isolated GB1 domain polypeptide of claim 3, further
comprising a mutation at a "knobs-into-holes" binding site for a Fc
fragment of an IgG, the mutation comprising an amino acid
substitution.
5. The isolated GB1 domain polypeptide of claim 4, wherein the
amino acid substitution comprises a comparatively non-polar amino
acid residue in place of a polar amino acid residue.
6. The isolated GB1 domain polypeptide of claim 4, further
comprising a mutation at the glutamate 27 residue of a native GB1
domain polypeptide, the mutation comprising an amino acid
substitution of the glutamate 27 residue.
7. The isolated GB1 domain polypeptide of claim 6, having an amino
acid sequence essentially as set forth in any SEQ ID NO:6, 20, 22
and 24.
8. The isolated GB1 domain polypeptide of claim 4, further
comprising a mutation at a lysine 28 residue of a native GB1 domain
polypeptide, the mutation comprising an amino acid substitution of
the lysine 28 residue.
9. The isolated GB1 domain polypeptide of claim 8, wherein the
mutation comprises substitution of the lysine 28 residue with a
comparatively non-polar amino acid residue.
10. The isolated GB1 domain polypeptide of claim 9, wherein the
non-polar amino acid residue is selected from the group consisting
of alanine, valine, leucine and isoleucine.
11. The isolated GB1 domain polypeptide of claim 10, having an
amino acid sequence essentially as set forth in SEQ ID NO:8.
12. The isolated GB1 domain polypeptide of claim 4, further
comprising a mutation at a lysine 31 residue of a native GB1 domain
polypeptide, the mutation comprising an amino acid substitution of
the lysine 31 residue.
13. The isolated GB1 domain polypeptide of claim 12, wherein the
mutation comprises substitution of the lysine 31 residue with a
comparatively non-polar amino acid residue.
14. The isolated GB1 domain polypeptide of claim 13, wherein the
non-polar amino acid residue is selected from the group consisting
of alanine, valine, leucine and isoleucine.
15. The isolated GB1 domain polypeptide of claim 14, having an
amino acid sequence essentially as set forth in SEQ ID NO:10.
16. The isolated GB1 domain polypeptide of claim 4, further
comprising a mutation at an asparagine 35 residue of a native GB1
domain polypeptide, the mutation comprising an amino acid
substitution of the asparagine 35 residue.
17. The isolated GB1 domain polypeptide of claim 16, wherein the
mutation comprises substitution of the asparagine 35 residue with a
comparatively non-polar amino acid residue.
18. The isolated GB1 domain polypeptide of claim 17, wherein the
non-polar amino acid residue is selected from the group consisting
of alanine, valine, leucine and isoleucine.
19. The isolated GB1 domain polypeptide of claim 18, having an
amino acid sequence essentially as set forth in SEQ ID NO:12.
20. The isolated GB1 domain polypeptide of claim 4, further
comprising a mutation at a tryptophan 43 residue of a native GB1
domain polypeptide, the mutation comprising an amino acid
substitution of the tryptophan 43 residue.
21. The isolated GB1 domain polypeptide of claim 20, wherein the
mutation comprises substitution of the tryptophan 43 residue with a
comparatively non-polar amino acid residue.
22. The isolated GB1 domain polypeptide of claim 21, wherein the
non-polar amino acid residue is selected from the group consisting
of alanine, valine, leucine and isoleucine.
23. The isolated GB1 domain polypeptide of claim 22, having an
amino acid sequence essentially as set forth in SEQ ID NO:16.
24. The isolated GB1 domain polypeptide of claim 4, further
comprising mutations at a threonine 35 residue and at a tyrosine 45
residue of a native GB1 domain polypeptide, the mutation comprising
an amino acid substitution of the threonine 35 residue and of the
tyrosine 45 residue.
25. The isolated GB1 domain polypeptide of claim 24, wherein the
mutation comprises substitutions of the threonine 35 residue and
the tyrosine 45 residue with a comparatively non-polar amino acid
residue.
26. The isolated GB1 domain polypeptide of claim 25, wherein the
non-polar amino acid residue is selected from the group consisting
of alanine, valine, leucine and isoleucine.
27. The isolated GB1 domain polypeptide of claim 26, having an
amino acid sequence essentially as set forth in SEQ ID NO:18.
28. The isolated GB1 domain polypeptide of claim 1, further
characterized as immobilized to a solid phase support.
29. The GB1 domain polypeptide of claim 1, wherein the Fab and the
Fc fragments are from an IgG from a warm-blooded vertebrate.
30. The GB1 domain polypeptide of claim 29, wherein the Fab and the
Fc fragments are from an IgG from a mammal.
31. The GB1 domain polypeptide of claim 30, wherein the mammal is
selected from the group consisting of human, mouse, pig, rat, ape,
monkey, cat, guinea pig, cow, goat and horse.
32. An isolated nucleic acid molecule encoding a GB1 domain
polypeptide which binds a Fab fragment of an IgG but does not bind
a Fc fragment of an IgG.
33. The isolated nucleic acid molecule of claim 32, wherein the
encoded polypeptide comprises an amino acid sequence selected from
among SEQ ID NO's:6, 8, 10, 12, 14, 16, 18, 20, 22 and 24.
34. The isolated nucleic acid molecule of claim 33, further defined
as comprising a GB1 domain polypeptide-encoding nucleic acid
molecule selected from among SEQ ID NO's:5, 7, 9, 11, 15, 17, 19,
21 and 23.
35. The isolated nucleic acid molecule of claim 32, further defined
as a DNA segment.
36. The isolated nucleic acid molecule of claim 32, further defined
as positioned under the control of a promoter.
37. The isolated nucleic acid molecule of claim 32, further defined
as a recombinant vector.
38. The isolated nucleic acid molecule of claim 37, wherein the
vector is a recombinant expression vector.
39. A recombinant host cell comprising the isolated nucleic acid
molecule of claim 32.
40. The recombinant host cell of claim 39, wherein the host cell is
a procaryotic cell.
41. The recombinant host cell of claim 39, wherein the host cell is
a eukaryotic cell.
42. A method of preparing a GB1 domain polypeptide which binds a
Fab fragment of an IgG but does not bind a Fc fragment of an IgG,
comprising: transforming a cell with isolated nucleic acid molecule
of claim 32 to produce a GB1 domain polypeptide which binds a Fab
fragment of an IgG but does not bind a Fc fragment of an IgG under
conditions suitable for the expression of the polypeptide.
43. A method for purifying Fc fragments of IgG's by affinity
chromatography, the method comprising the steps of: (a) contacting
a sample comprising IgG Fc and Fab fragments with a GB1 polypeptide
of claim 1, the GB1 domain polypeptide immobilized to a solid phase
support, to immobilize the IgG Fab fragments to the solid phase
support; and (b) collecting the IgG Fc fragment remaining in the
sample.
44. The method of claim 43, wherein the Fab and the Fc fragments
are from an IgG from a warm-blooded vertebrate.
45. The method of claim 44, wherein the Fab and the Fc fragments
are from an IgG from a mammal.
46. The method of claim 45, wherein the mammal is selected from the
group consisting of human, mouse, pig, rat, ape, monkey, cat,
guinea pig, cow, goat and horse.
47. A method for purifying Fab fragments of IgG's by affinity
chromatography, the method comprising the steps of: (a) contacting
a sample comprising IgG Fc and Fab fragments with a GB1 polypeptide
of claim 1, the GB1. polypeptide immobilized to a solid phase
support, to immobilize the IgG Fab fragments to the solid phase
support; (b) collecting the IgG Fc fragment remaining in the
sample; and (c) eluting the IgG Fab fragments from the solid phase
support to give purified IgG Fab fragments in the eluate.
48. The method of claim 47, wherein the IgG Fab fragments bound to
the immobilized GB1 polypeptide are eluted by washing the solid
phase support with a buffer of about pH 3.5 to about pH 2.4 to give
the Fab fragments in the eluate.
49. The method of claim 47, wherein the Fab and the Fc fragments
are from an IgG from a warm-blooded vertebrate.
50. The method of claim 49, wherein the Fab and the Fc fragments
are from an IgG from a mammal.
51. The method of claim 50, wherein the mammal is selected from the
group consisting of human, mouse, pig, rat, ape, monkey, cat,
guinea pig, cow, goat and horse.
52. A method for detecting IgG, a fragment of an IgG, or
combinations thereof, in a fluid sample suspected of containing
IgG, a fragment of an IgG, or combinations thereof, the method
comprising the steps of: (a) contacting the fluid sample with a
binding substance comprising the GB1 polypeptide of claim 1, under
conditions favorable to binding of IgG, a fragment of an IgG, or
combinations thereof to the binding substance to form a complex
therebetween; and (b) detecting the complex by means of a label
conjugated to the binding substance or by means of a labeled
reagent that specifically binds to the complex subsequent to its
formation.
53. The method of claim 52, wherein the binding substance is
conjugated with a detectable label and wherein detecting step (b)
comprises: i) separating the complex from unbound labeled binding
substance; and ii) detecting the detectable label which is present
in the complex or which is unbound.
54. The method of claim 53, wherein the fragments of the IgG are
Fab and the Fc fragments are from an IgG from a warm-blooded
vertebrate.
55. The method of claim 54, wherein the Fab and the Fc fragments
are from an IgG from a mammal.
56. The method of claim 55, wherein the mammal is selected from the
group consisting of human, mouse, pig, rat, ape, monkey, cat,
guinea pig, cow, goat and horse.
Description
TECHNICAL FIELD
[0002] The present invention relates to detection and purification
of antibody fragments. More particularly, the present invention
relates to the detection and purification of Fc and Fab fragments
of IgG's using reagents prepared from the B1 domain of bacterial
protein G.
1 Table of Abbreviations ELISA enzyme-linked immunosorbent assay
Fab antigen binding fragment of an immunoglobulin Fc readily
crystallized fragment of an immunoglobulin GB1 B1 domain of Protein
G HFc readily crystallized fragment of a human immunoglobulin Ig
immunoglobulin IgG immunoglobulin G PCR polymerase chain reaction
pfu plaque forming units
BACKGROUND ART
[0003] Protein-protein interactions play an essential role in many
biological processes. Understanding the energetics of such
interactions is of great importance because it defines the
necessary concentrations of interacting partners, the rates at
which these partners are capable of associating, and the relative
concentrations of bound and free proteins in a solution. See
Stites. W. E. (1997) Chem. Rev. 97:1233-1250. Well studied classes
of protein-protein interactions (Jones. S. and Thornton. J. M.
(1996) Proc. Natl. Acad. Sci. USA 93:13-20; LeConte. L. et al.
(1999) J. Mol. Biol. 285:2177-2198) include hormone receptor
binding and activation (Wells. J. A. and deVos. A. M. (1996) Ann.
Rev. Biochem. 65:609-634), antibodies with protein antigens
(Davies, D. R. and Cohen, G. H. (1996) Proc. Natl. Acad. Sci. USA
93:7-12), enzyme inhibitor complexes (Tsunogae, Y. et al. (1986) J.
Biochem. 100:1637-46), and protein oligomerization (Argos, P.
(1988) Prot. Eng. 2:101-113).
[0004] B1 is one of the domains of Protein G, a member of an
important class of proteins which form IgG-binding receptors on the
surface of certain staphylococcal and streptococcal strains, as
described by Boyle. M. D. P. (1990) Bacterial
Immunoglobulin-Binding Proteins, Academic Press, San Diego and
Frick, I-M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:8532-8536).
It has been suggested that these proteins allow the pathogenic
bacterium to evade the host immune response by coating the invading
bacteria with host antibodies (Goward. C. R. et al. (1993) Trends
Biochem. Sci. 18:136-140), thereby contributing significantly to
the pathogenicity of these bacteria. Furthermore, protein G has
found numerous applications in biotechnology as a reagent for
affinity purification of antibodies (Stahl, S. et al. (1993)
Current Opinion in Immunology 5:272-277), since it binds to IgGs of
many different species and subclasses, as disclosed in Stone. G. C.
et al. (1989) J. Immunol 143:565-570 and in Fahnestock et al.
(1990) U.S. Pat. No. 4,977,247. Further characterization of the
sequence determinants that contribute to IgG binding may lead to
new therapeutics for streptococcal infections and novel
immunochemical reagents and thus represents a significant need in
the art.
[0005] Staphylococcal Protein A competitively binds to a similar
site on the Fc fragment of human IgG's as the B1 domain, involving
in both cases hFc residues 252-254, 433-435, and 311, as described
by Deisenhofer, J. (1981) Biochemistry 20:2361-2370 and
Sauer-Eriksson, A. E. et al. (1995) Structure 3:265-278. Whereas
the interactions between B1 and hFc are predominantly polar, half
of the protein A interactions are polar and half are hydrophobic.
These proteins present an example of two distinct folds which have
evolved different structural features to achieve binding at very
similar sites on the same target molecule.
[0006] The B1 domain of Protein G is a 56-residue domain that folds
into a four-stranded .beta.-sheet and one .alpha.-helix, as shown
by NMR and X-ray crystallography. Despite its small size, the B1
domain has two separate IgG-binding sites on its surface, each
interacting respectively with specific, independent sites on the
Fab or Fc fragments of the antibody. Compared to most other
protein-protein interactions, the Fc-binding site on the B1 protein
is somewhat a typical. First, it is predominantly polar rather than
hydrophobic in character (Stites. W. E. (1997) Chem. Rev.
97:1233-1250); second, the interfacial area is on the lower end of
the observed range, -700 .ANG..sup.2 rather than the average 1200
.ANG..sup.2 (Jones, S. and Thornton, J. M. (1996) Proc. Natl. Acad.
Sci. USA 93:13-20); third, rather than a planar interaction surface
typically observed in heterodimers, this interface is formed by a
double "knobs-into-holes" interaction (Crick. F. H. C. (1952)
Nature 170:882-883; Crick. F. H. C. (1953) Acta Crystallographica
6:689-697) in which a knob from the B1 protrudes into a hole in the
hFc, and vice versa.
[0007] In view of the presence of these a typical elements within
the Fc-binding site on the B1 domain of protein G, the
characterization of the energetic contributions of each of these
elements on the B1 domain for Fc fragments of IgG's represents a
long-felt and significant need in the art. Indeed, the
characterization of the energetic contributions of the a typical
elements within the Fc-binding site on the B1 protein would
facilitate the development of improved reagents and methods for
detection and purification of antibody fragments, among other
applications. The development of such reagents and methods thus
represents an ongoing need in the art.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, an isolated GB1
domain polypeptide which exhibits binding activity for an Fab
fragment of an IgG but exhibits substantially no binding activity
for an Fc fragment of an IgG is disclosed.
[0009] Preferably, the isolated GB1 domain polypeptide of the
present invention further comprises a disrupted "knobs-into-holes"
binding site for a Fc fragment of an IgG. More preferably still,
the isolated GB1 domain polypeptide of the present invention
further comprises a mutation at a "knobs-into-holes" binding site
on the GB1 polypeptide for a Fc fragment of an IgG GB1 domain
polypeptide, the mutation comprising an amino acid
substitution.
[0010] In accordance with the present invention, a method for
purifying Fc fragments of IgG's by affinity chromatography is also
disclosed. The method comprising the steps of: (a) contacting a
sample comprising IgG Fc and Fab fragments with a GB1 polypeptide
of the present invention immobilized to a solid phase support to
immobilize the IgG Fab fragments to the solid phase support; and
(b) collecting the IgG Fc fragment remaining in the sample.
[0011] In accordance with the present invention, a method for
purifying Fab fragments of IgG's by affinity chromatography is also
disclosed. The method comprising the steps of: (a) contacting a
sample comprising IgG Fc and Fab fragments with a GB1 polypeptide
of the present invention immobilized to a solid phase support to
immobilize the IgG Fab fragments to the solid phase support; (b)
collecting the IgG Fc fragment remaining in the sample; and (c)
eluting the IgG Fab fragments from the solid phase support to give
purified IgG Fab fragments in the eluate.
[0012] In accordance with the present invention, a method for
detecting IgG, a fragment of an IgG, or combinations thereof, in a
fluid sample suspected of containing IgG, a fragment of an IgG, or
combinations thereof is also disclosed. The method comprising the
steps of: (a) contacting the fluid sample with a binding substance
comprising the GB1 polypeptide of claim 1, under conditions
favorable to binding of IgG, a fragment of an IgG, or combinations
thereof to the binding substance to form a complex therebetween;
and (b) detecting the complex by means of a label conjugated to the
binding substance or by means of a labeled reagent that
specifically binds to the complex subsequent to its formation.
[0013] It is thus an object of the present invention to provide
novel reagents and methods for the detection and purification of
antibody fragments.
[0014] It is another object of the present invention to novel
reagents and methods for the detection and purification of antibody
fragments which provides for the separation of Fc fragments and Fab
fragments of an IgG, preferably an IgG of a warm-blooded
vertebrate.
[0015] It is yet another object of the present invention to novel
reagents and methods for the detection and purification of antibody
fragments which provides for the separation of Fc fragments and Fab
fragments of a human IgG.
[0016] It is still another object of the present invention to
provide for the characterization of the energetic contributions of
elements within the Fc-binding site on the B1 domain of protein G
to binding with an Fc fragment of an IgG, preferably an IgG of a
warm-blooded vertebrate.
[0017] Some of the objects of the invention having been stated
hereinabove, other objects will become evident as the description
proceeds, when taken in connection with the accompanying drawings
and Examples as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 presents three schematic depictions of the
interaction between the B1 domain and a human Fc fragment. All
structures were drawn from the coordinates of the B1-hFc complex
described by Sauer-Eriksson et al. (1995) Structure 3:265-278 and
at Brookhaven accession number lfcc.
[0019] FIG. 1A is a ribbon representation with the two partners
pried apart. The residues on the surface of the B1 domain are
colored according to the loss in binding free energy when mutated
to alanine: yellow, >500-fold, orange,>300-fold; red,
>50-fold, brown,>10-fold; grey,<2-fold. The approximate
position of the reporter fluofophore covalently attached to a
cysteine at position 32 is indicated by the purple sphere.
[0020] FIG. 1B is a surface rendering of the interaction,
highlighting the position of the knobs and holes on the two
proteins.
[0021] FIG. 1C depicts a stereopair showing details of the
interaction between the B1 domain (heavy lines) and the Cy2-Cy3
region of the Fc fragment (light lines).
[0022] FIG. 2 presents two graphs depicting the interaction of B1
mutants with a human Fc fragment. Binding was monitored by changes
in fluorescence of an acrylodan reporter group site-specifically
attached to a cysteine mutation at position 32 of the B1 domain.
Each point represents the average of three independent titrations,
with experimental errors as indicated.
[0023] FIG. 2A is a line graph depicting titration of the unlabeled
Fc fragment into a solution of 250 nM of the T25A mutant B1 domain.
The data was fit to Equation 1. Experimental Error bars are smaller
than symbols on graph.
[0024] FIG. 2B is a line graph depicting determination of the free
energy of binding of a weakly binding B1 mutant, N35A, by a
competition experiment in which unlabeled mutant protein is
titrated into a 250 nM preformed complex of fluorescent wild-type
B1 and human Fc. The data was fit to Equation 1.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In accordance with the present invention, the contribution
to the free energy of binding of each of the residues forming the
binding site for an IgG Fc fragment on the surface of the B1 domain
of protein G was determined by alanine-scanning mutagenesis. The
interface between these two proteins is a typical in that it is
smaller than usual, polar in character and involves two
well-defined "knobs-into-holes" interactions. The bulk of the free
energy of binding is contributed by three central residues which
make hydrogen bonds across the interface.
[0026] Of these, the most critical interaction is formed by the
glutamate 27 (Glu27) residue, which acts as a charged knob on the
surface of the B1 domain, inserting into a polar hole on the Fc
fragment. A single alanine mutation of this residue virtually
abolishes stable complex formation. Indeed, it was observed that
the substitution of the Glu 27 residue with any of the other
nineteen amino acids virtually abolishes stable complex formation.
Formation of a stable interface between these two proteins is
therefore dominated by a small, polar "hot-spot". Thus, the
detection and purification reagents and methods of the present
invention in part pertain to disruption of the small, polar "hot
spot".
[0027] A. General Considerations
[0028] B1 is one of the domains of Protein G, a member of an
important class of proteins which form IgG-binding receptors on the
surface of certain staphylococcal and streptococcal strains. See
Boyle. M. D. P. (1990) Bacterial Immunoglobulin-Binding Proteins,
Academic Press, San Diego, Calif. and Frick. I-M. et al. (1992)
Proc. Natl. Acad. Sci. USA 89:8532-8536. The B1 domain has found
numerous applications in biotechnology as a reagent for qffinity
purification of antibodies (Stahl, S. et al. (1993) Current Opinion
in Immunology 5:272-277), since it binds to IgGs of many different
species and subclasses as disclosed by Stone. G. C. et al. (1989)
J. Immunol. 143:565-570.
[0029] B1 is a 56-residue domain that folds into a four-stranded
.beta.-sheet and one .alpha.-helix, as shown by NMR and X-ray
crystallography. Despite its small size, the B1 domain has two
separate IgG-binding sites on its surface, each interacting
respectively with specific, independent sites on the Fab or Fc
fragments of the antibody. The structure of a B1-Fab complex
(Derrick. J. P. and Wigley, D. B. (1992) Nature 359:752-754 has
revealed that the Fab-binding site is mediated almost entirely
through backbone contacts between the edge of the .alpha.-sheet of
the B1 domain and the last .beta.-strand of the C.sub.H1 domain of
the Fab fragment, thereby forming a continuous .beta.-sheet
spanning the interface between the two partners.
[0030] In contrast, a B1-Fc complex (Sauer-Eriksson, A. E. et al.
(1995) Structure 3: 265-278) has shown that the Fc-binding site is
mediated primarily by side-chain contacts between the two proteins.
This is further supported by competition experiments in which an
11-residue peptide corresponding to the C-terminus of the
.alpha.-helix and the N-terminal part of the third .beta.-strand
competitively inhibits binding of B1 to hFc. Frick, I-M. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:8532-8536. NMR experiments
also confirm the general location of the Fc-binding site on the
surface of the B1 domain (Gronenborn. A. M. and Clore. G. M. (1993)
J. Mol. Biol. 233:331-335; Kato, K. et al. (1995) Structure
3:79-85). However, prior to the disclosure of the present
application, the energetics of B1-Fc binding have not been
characterized.
[0031] The term "antibody or antibody molecule" in the various
grammatical forms is used herein as a collective noun that refers
to a population of immunoglobulin molecules and/or immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antibody combining site or paratope. An "antibody
combining site" is that structural portion of an antibody molecule
comprised of heavy and light chain variable and hypervariable
regions that specifically binds antigen. Antibodies, including
polyclonal and monoclonal antibodies, can be prepared in accordance
with a variety of art-recognized techniques, such as are
exemplified in Howell et al. (1988) Antibodies A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0032] Five classes of immunoglobulin have been defined in humans
and the higher mammals. Those are IgG, IgM, IgA, IgD, and IgE.
Additionally, humans have been found to have four subclasses of IgG
and two subclasses of IgA. These immunoglobulins are present in all
normal individuals and are referred to as isotypes. The type of
heavy chain involved, termed gamma (y), mu (p), alpha (.alpha.),
delta (.delta.), and epsilon (.epsilon.), respectively, establishes
the class of immunoglobulin. Each isotype is characterized by its
amino acid sequence and is the product of a different gene segment.
Additionally, two types of human immunoglobulin light chain were
also defined by their distinct antigenicity and named kappa
(.kappa.) and lambda (.lambda.).
[0033] When an Ig molecule is digested by papain to yield
fragments, and these digestion products are dialyzed, protein
crystals accounting for one-third of the original protein mass are
produced. These crystals are termed the Fc fragment (the complement
binding fragment) as they constitute the "fragment crystallizable".
Fc, comprising the carboxy-termini of two heavy chains, is dimeric
in nature. The heavy chains are held together by inter-chain
disulfide bonds. In addition, intra-chain disulfide bonds add to
the conformation of the Fc region. Carbohydrates are found attached
to the Fc portion of immunoglobulin.
[0034] The fragments which account for two-thirds of the original
protein mass after papain digest of an Ig bind antigen in a manner
equivalent to the original molecule and are termed the Fab
fragments as they were antigen binding. Fab fragments are also
known as monovalent fragments. It is often desirable to isolate Fab
fragments from Fc fragments to study biological characteristics,
such as binding characteristics, of the separate fragments. Indeed,
papain digestion yielding Fab monomers almost always generates Fc
fragments and as such, the presence of Fc fragments is always an
issue if the Fab monomer is the desired product of the digest. The
present invention provides methods and reagents useful in isolation
of the fragments and thus solves a particular problem associated
with papain digestion.
[0035] Alternatively, antibody fragments may be prepared by pepsin
cleavage, which releases a bivalent antigen-binding F(ab').sub.2
fragment and a complement-binding Fc' fragment. The F(ab').sub.2
fragment can be dissociated by thiol reagents into monovalent
fragments. The present invention also provides methods and reagents
useful in isolation of F(ab').sub.2 fragments.
[0036] The present invention particularly concerns IgG's and
fragments thereof, including particularly the papain digestion
fragments Fab and Fc. Thus, as sed herein and in the claims, the
term "IgG" is meant to refer to any IgG, including but not limited
to IgG's from any warm-blooded vertebrate subject, and including
but not limited to polyclonal IgG's and monoclonal IgG's. As is
recognized among those having ordinary skill in the art, IgG's,
including warm-blooded vertebrate IgG's, may be divided into
subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4.
Thus, the term "IgG" is also meant to include all subclasses of
IgG's.
[0037] The recognized binding of protein G and the B1 domain of
protein G with IgG's of many different species and subclasses, as
disclosed in Stone, G. C. et al. (1989) J. Immunol. 143:565-570
coupled with the sequence modification techniques, peptide
synthesis techniques, and laboratory examples disclosed herein
provide adequate guidance to one of ordinary skill in the art for
the detection and purification of IgG's and fragments thereof,
including Fab and Fc fragments, from any warm-blooded vertebrate
species using the GB1 polypeptides of the present invention in
accordance with the detection and isolation methods disclosed
herein.
[0038] As disclosed in detailed herein, the Fc binding segment of
the GB1 polypeptide of the present invention has been modified to
disrupt binding. As shown below in Table 1, mammalian Fc fragments
include highly homologous structures as compared to human IgG Fc
fragments, including substantially invariant residues at a binding
site on the Fc fragment for a native or naturally occurring GB1
domain polypeptide. Thus, a GB1 domain polypeptide of the present
invention having binding activity towards an IgG Fab fragment from
a warm-blooded vertebrate, preferably a mammal, and having
substantially no binding activity towards a mouse IgG Fc fragment a
warm-blooded vertebrate, preferably a mammal, may be prepared using
the sequence modification data and techniques, and protein
synthesis techniques, disclosed herein. Therefore, a "GB1 domain
polypeptide of the present invention" as used herein and in the
claims is meant to encompass such a polypeptide.
[0039] Table 1 presents a compilation of a sequence alignment data
for preferred IgG species and subtypes. This alignment was
constructed through a blast search which was performed on the World
Wide Web at: http://www.ncbi.nlm.nih.gov/BLAST/. The target
sequence that was searched with was a generic eukaryotic IgG Fc
sequence and these were then all aligned against a human IgG1 Fc
sequence. The alignment shown in Table 1 below is of the most
relevant regions of the Fc domain which contacts with the B1
domain. See also FIG. 1B. In each segment of 10 residues, it is the
middle two residues (residue number 5 and 6, I and S in the first
column and H and N in the second column) that are deemed to be
important Fc residues for binding. It is noted that these residues
are nearly invariant. The numbers shown are the amino acid number
for the four important residues, the numbering convention is that
from human IgG1 Fc fragment.
2 TABLE 1 253, 254 433, 434 IgG k chain DTLMISRTPE HEALHNHYTQ IgG1
human DTLMISRTPE HEALHNHYTQ IgG2 human DTLMISRTPE HEALHNHYTQ IgG3
human DTLMISRPTE HEALHNRFTQ IgG4 human DTLMISRTPE HEALHNHYTQ IgG1
mouse DVLTITLTPK HEGLHNHHTE IgG2a mouse DVLMISLSPI HEGLHNHHTT IgG2b
mouse DVLMISLTPK HEGLKNYYLK IgG3 mouse DALMISLTPK HEALHNHHTQ IgG2a
pig DTLMISRTPQ HEALHNHYTQ IgG2b pig DTLMISRTPQ HEALHNHYTQ IgG3 pig
DTLMISQTPE HEALHNHYTQ IgG4 pig DTLMISRTPK HEALHNHYTQ IgG Rat
DTLMISRTPE HEALHNHYTQ IgG chimp DTLMISRTPE HEALHNHYTQ IgG Macaca
DTLMISRTPE HEALHNHYTQ IgG rabbit DTLMISRTPE HEALHNHYTQ IgG1 cat
DTLSISRTPE HEALHSHHTQ IgG2 guinea pig DTLMISLTPR HEALHNHVTQ IgG3
cow DTLTISGTPE HEALRNHYKE
[0040] Thus, the GB1 polypeptides of the present invention may be
used to bind IgG's from any suitable or desirable warm-blooded
vertebrate species, including mammalian and avian species. In most
research or clinical applications, the more commonly used
polyclonal antibodies are likely to be prepared and isolated from
rabbit, goat and horse, while the more commonly used monoclonal
antibodies are likely to be prepared and isolated from mouse. IgG's
and fragments thereof, including Fab and Fc fragments, prepared and
isolated from mouse, rat, rabbit, goat, human and horse are
particularly contemplated in accordance with the present
invention.
[0041] For example, Fab and Fc fragments from a mouse IgG antibody,
particularly a monoclonal antibody, have been purified and
separated by the inventors in accordance with the techniques
described herein using a GB1 polypeptide of the present invention.
Fab and Fc fragments of IgG antibodies from goat and rabbit have
also been purified and isolated using a GB1 polypeptide of the
present invention. Therefore, the phrases "having binding activity
for an IgG Fab fragment" and "having substantially no binding
activity towards an IgG Fc fragment" as used herein in connection a
GB1 polypeptide of the present invention are meant to encompass
such binding characteristics as applied to IgG's, and fragments
thereof, from any warm-blooded vertebrate species. Preferably,
these phrases are meant to encompass IgG's, and fragments thereof,
from mammalian species. Preferred mammalian species include are
listed in Table 1 above, and include human, mouse, pig, rat, ape,
monkey, cat, guinea pig, cow, goat and horse.
[0042] B. Polypeptides
[0043] In accordance with the present invention, an isolated GB1
domain polypeptide which exhibits binding activity for an Fab
fragment of an IgG but exhibits substantially no binding activity
for an Fc fragment of an IgG is disclosed. The terms "bind",
"binding", "binding activity" and "binding affinity" are believed
to have well-understood meanings in the art. To facilitate
explanation of the present invention, the terms "bind" and
"binding" are meant to refer to protein-protein interactions that
are recognized to play an essential role in many biological
processes, such as the binding between an antibody and an antigen.
Exemplary protein-protein interactions include, but are not limited
to, covalent interactions between side chains, such as disulfide
bridges between cysteine residues; hydrophobic interactions between
side chains; and hydrogen bonding between side chains. Particularly
contemplated protein-protein interactions for the present invention
are the hydrogen bonds formed between a polar "hot spot" of the
natural B1 domain polypeptide and the Fc fragment of an IgG.
[0044] The terms "binding activity" and "binding affinity" are
meant to refer to the tendency of one protein or polypeptide to
bind or not to bind to another protein or polypeptide. The
energetics of protein-protein interactions are significant in
"binding activity" and "binding affinity" because they define the
necessary concentrations of interacting partners, the rates at
which these partners are capable of associating, and the relative
concentrations of bound and free proteins in a solution.
[0045] Thus, as used herein and in the claims, the terms
"substantially no binding activity" or "substantially no binding
affinity" refer to a substantial lack of binding or lack of
interaction between two polypeptides, e.g. between the Fc fragment
of an IgG and a GB1 domain polypeptide of the present invention.
These terms can be further quantified from the detailed energetics
data presented in the Examples with respect to binding and lack of
binding between polypeptides of the present invention and Fc
fragments of IgG's. Thus, for example, a dissociation constant
(K.sub.d) may be used to describe a GB1 polypeptide of the present
invention having "substantially no binding activity" for an Fc
fragment of an IgG. Indeed, preferably, an isolated GB1 domain
polypeptide of the present invention which exhibits substantially
no binding activity for an Fc fragment of an IgG is characterized
as having a disassociation constant for a Fc fragment of an IgG of
greater than about 2 mM.
[0046] More preferably, the isolated GB1 domain polypeptide of the
present invention further comprises a disrupted "knobs-into-holes"
binding site for a Fc fragment of an IgG. More preferably still,
the isolated GB1 domain polypeptide of the present invention
further comprises a mutation at a "knobs-into-holes" binding site
on the GB1 polypeptide for a Fc fragment of an IgG, the mutation
comprising an amino acid substitution. Optionally, the amino acid
substitution comprises the substitution of a polar amino acid
residue with a comparatively non-polar amino acid residue.
Preferably, the Fc is an Fc fragment of an IgG of a warm-blooded
vertebrate, such as mouse, rabbit, goat, horse or human. Fc
fragments of a human IgG are also referred to herein as an "hFc
fragment".
[0047] A preferred embodiment of a GB1 domain polypeptide of the
present invention comprises a mutation at the glutamate 27 residue
of a native GB1 domain polypeptide. The mutation comprises a
substitution of the glutamate 27 residue with any of the other 19
amino acids, as it has been observed by the present inventors that
any such substitution substantially abolishes Fc binding activity
while maintaining Fab binding activity. Optionally, the
substitution may comprise a comparatively non-polar amino acid
residue, such as alanine, valine, leucine and isoleucine. In the
case of an hFc fragment, the mutation is further characterized as a
substitution of the glutamate 27 residue with a residue
substantially incapable of forming a hydrogen bond with an O.sub.y
of a serine 254 residue of the hFc fragment. Particularly
contemplated examples of such a GB1 polypeptide are disclosed in
SEQ ID NO's: 6, 20, 22 and 24.
[0048] An alternative embodiment of a GB1 domain polypeptide of the
present invention comprises a mutation at a lysine 28 residue of a
native GB1 domain polypeptide. The mutation comprises a
substitution of the lysine 28 residue with any of the other 19
amino acids, as it is contemplated by the present inventors that
any such substitution substantially abolishes Fc binding activity
while maintaining Fab binding activity. Optionally, the
substitution may comprise a comparatively non-polar amino acid
residue, such as alanine, valine, leucine and isoleucine. A
particularly contemplated example of such a GB1 polypeptide is
disclosed in SEQ ID NO: 8.
[0049] An alternative embodiment of a GB1 domain polypeptide of the
present invention comprises a mutation at a lysine 31 residue of a
native GB1 domain polypeptide. The mutation comprises a
substitution of the lysine 31 residue with any of the other 19
amino acids, as it is contemplated by the present inventors that
any such substitution substantially abolishes Fc binding activity
while maintaining Fab binding activity. Optionally, the
substitution may comprise a comparatively non-polar amino acid
residue, such as alanine, valine, leucine and isoleucine. A
particularly contemplated example of such a GB1 polypeptide is
disclosed in SEQ ID NO:10.
[0050] An alternative embodiment of a GB 1 domain polypeptide of
the present invention comprises a mutation at an asparagine 35
residue of a native GB1 domain polypeptide. The mutation comprises
a substitution of the asparagine 35 residue with any of the other
19 amino acids, as it is contemplated by the present inventors that
any such substitution substantially abolishes Fc binding activity
while maintaining Fab binding activity. Optionally, the
substitution may comprise a comparatively non-polar amino acid
residue, such as alanine, valine, leucine and isoleucine. A
particularly contemplated example of such a GB1 polypeptide is
disclosed in SEQ ID NO:12.
[0051] An alternative embodiment of a GB1 domain polypeptide of the
present invention comprises a mutation at a tryptophan 43 residue
of a native GB1 domain polypeptide. The mutation comprises a
substitution of the tryptophan 43 residue with any of the other 19
amino acids, as it is contemplated by the present inventors that
any such substitution substantially abolishes Fc binding activity
while maintaining Fab binding activity. Optionally, the
substitution may comprise a comparatively non-polar amino acid
residue, such as alanine, valine, leucine and isoleucine. A
particularly contemplated example of such a GB1 polypeptide is
disclosed in SEQ ID NO:16.
[0052] An alternative embodiment of a GB1 domain polypeptide of the
present invention comprises mutations at a threonine 35 residue and
at a tyrosine 45 residue of a native GB1 domain polypeptide. The
mutation comprises a substitution of the threonine 35 residue and
tyrosine 45 residues with any of the other 19 amino acids, as it is
contemplated by the present inventors that any such substitution
substantially abolishes Fc binding activity while maintaining Fab
binding activity. Optionally, the substitution may comprise a
comparatively non-polar amino acid residue, such as alanine,
valine, leucine and isoleucine. A particularly contemplated example
of such a GB1 polypeptide is disclosed in SEQ ID NO:18.
[0053] Thus, as used herein and in the claims, the terms "GB1
domain protein" and "GB1 domain polypeptide" refer to polypeptides
having amino acid sequences which are substantially identical to
the naturally occurring or native amino acid sequences in the GB1
domain but which are altered, mutated or otherwise changed so as to
exhibit Fab binding activity and substantially no Fc binding
activity.
[0054] The terms "GB1 domain protein" and "GB1 domain polypeptide"
also include analogs of GB1 domain molecules of the present
invention which exhibit at least some biological activity in common
with native GB1 domain polypeptides, that is Fab binding activity,
while at the same time exhibiting substantially no Fc binding
activity in accordance with the present invention. Furthermore,
those skilled in the art of mutagenesis will appreciate that other
analogs, as yet undisclosed or undiscovered, may be used to
construct GB1 domain analogs. There is no need for a "GB1 domain
protein" or "GB1 domain polypeptide" to comprise all, or
substantially all of the amino acid sequence of a native GB1 domain
polypeptide. Shorter or longer sequences are anticipated to be of
use in the invention. Thus, these terms also include fusion or
recombinant GB1 domain polypeptides and proteins. Methods of
preparing such proteins are described herein in the Examples among
other places.
[0055] The terms "GB1 domain-encoding nucleic acid sequence" and
"GB1 domain-encoding nucleic acid segment" refer to any DNA
sequence that is substantially identical to a polynucleotide
sequence encoding a GB1 domain protein or GB1 domain polypeptide as
defined above. The terms also refer to RNA, or antisense sequences,
compatible with such DNA sequences. A "GB1 domain-encoding nucleic
acid sequence" and "GB1 domain-encoding nucleic acid segment" may
also comprise any combination of associated control sequences.
[0056] The term "substantially identical", when used to define
either a GB1 domain polypeptide amino acid sequence, or a GB1
domain polypeptide-encoding nucleic acid sequence, means that a
particular sequence, for example, a mutant sequence, varies from
the sequence of a GB1 domain polypeptide of the present invention
or a native GB1 domain polypeptide by one or more deletions,
substitutions, or additions, the net effect of which is to retain
at least some of a desired binding activity or other biological
activity of the GB1 domain polypeptide. Alternatively, DNA analog
sequences are "substantially identical" to specific DNA sequences
disclosed herein if: (a) the DNA analog sequence is derived from
coding regions of a natural GB1 domain-encoding nucleic acid
molecule; or (b) the DNA analog sequence is capable of
hybridization of DNA sequences of (a) under moderately stringent
conditions and which encode a GB1 domain polypeptide as defined
herein above; or (c) the DNA sequences are degenerative as a result
of the genetic code to the DNA analog sequences defined in (a)
and/or (b).
[0057] Substantially identical analog proteins preferably will be
greater than about 60% identical to the corresponding sequence of a
particular sequence of sequence of a GB1 domain polypeptide of the
present invention disclosed herein, or of the native GB1 protein.
Sequences having lesser degrees of similarity but comparable
binding activity are considered to be equivalents. In determining
nucleic acid sequences, all subject nucleic acid sequences capable
of encoding substantially similar amino acid sequences are
considered to be substantially similar to a reference nucleic acid
sequence, regardless of differences in codon sequences.
[0058] The GB1 polypeptides of the present invention exhibiting
binding activity for Fab fragments of IgG's, particularly
warm-blooded vertebrate IgG's, more particularly mammalian IgG's,
and even more particularly mouse, rat, goat, rabbit, human and
horse IgG's. Moreover, as discussed herein above, the Fab binding
domain of the GB1 domain has been characterized in the art. Thus,
any Fab binding domain of the GB1 domain having Fab binding
activity may be incorporated into a GB1 domain polypeptide of the
present invention, including preferably the Fab binding domain of
the native GB1 domain. Additionally, as described herein below, any
desirable biological functional equivalent mutation, substitution,
alteration or other change in the Fab binding domain of the GB1
domain is well within the skill of the art from the disclosure
herein and is also contemplated to fall within the scope of the
present invention.
[0059] C. Percent Similarity
[0060] Percent similarity may be determined, for example, by
comparing sequence information using the GAP computer program,
available from the University of Wisconsin Geneticist Computer
Group. The GAP program utilizes the alignment method of Needleman
et al. (1970), as revised by Smith et al. (1981). Briefly, the GAP
program defines similarity as the number of aligned symbols (i.e.
nucleotides or amino acids) which are similar, divided by the total
number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include: (1) a
unitary comparison matrix (containing a value of 1 for identities
and 0 for non-identities) of nucleotides and the weighted
comparison matrix of Gribskov et al. (1986), as described by
Schwartz et al. (1979); (2) a penalty of 3.0 for each gap and an
additional 0.01 penalty for each symbol and each gap; and (3) no
penalty for end gaps. Other Comparison techniques are described in
the Examples.
[0061] The term "homology" describes a mathematically based
comparison of sequence similarities which is used to identify genes
or proteins with similar functions or motifs. Accordingly, the term
"homology" is synonymous with the term "similarity" and "percent
similarity" as defined above. Thus, the phrases "substantial
homology" or "substantial similarity" have similar meanings.
[0062] D. Nucleic Acid Sequences
[0063] In certain embodiments, the invention concerns the use of
GB1 domain polypeptides and GB1 domain polypeptide-encoding nucleic
acids that include within their respective sequences a sequence
which is essentiallythat of a GB1 domain-encoding nucleic acid, or
the corresponding protein. The term "a sequence essentially as that
of a GB1 domain-encoding nucleic acid" means that the sequence
substantially corresponds to a portion of a GB1 domain polypeptide
or GB1 domain polypeptide-encoding nucleic acid and has relatively
few bases or amino acids (whether nucleic acid or protein) which
are not identical to those of a GB1 domain protein or GB1
domain-encoding nucleic acid, (or a biologically functional
equivalent of, when referring to proteins). The term "biologically
functional equivalent" is well understood in the art and is further
defined in detail herein. Accordingly, sequences which have between
about 70% and about 80%; or more preferably, between about 81% and
about 90%; or even more preferably, between about 91% and about
99%; of amino acids which are identical orfunctionally equivalent
to the amino acids of a GB1 domain protein or GB1 domain-encoding
nucleic acid, will be sequences which are "essentially the
same".
[0064] GB1 domain polypeptides and GB1 domain-encoding nucleic
acids which have functionally equivalent codons are also covered by
the invention. The term "functionally equivalent codon" is used
herein to refer to codons that encode the same amino acid, such as
the six codons for arginine or serine, and also to refer to codons
that encode biologically equivalent amino acids (see Table 2).
3TABLE 2 Functionally Equivalent Codons. Amino Acids Codons Alanine
Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D
GAC GAU Glumatic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine
Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA
CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline
Pro P CCA CCC CCG CCU Glutamine Gln Q CM CAG Arginine Arg R AGA AGG
OGA CCC CGG CGU Serine Ser S ACG AGU UCA UCC UCG UCU Threonine Thr
T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
[0065] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of IgG Fab fragment binding activity as
well as the substantial lack of binding activity for IgG Fc
fragments. The addition of terminal sequences particularly applies
to nucleic acid sequences which may, for example, include various
non-coding sequences flanking either of the 5' or 3' portions of
the coding region or may include various internal sequences, i.e.,
introns, which are known to occur within genes.
[0066] The present invention also encompasses the use of DNA
segments which are complementary, or essentially complementary, to
the sequences set forth in the specification. Nucleic acid
sequences which are "complementary" are those which are
base-pairing according to the standard Watson-Crick complementarity
rules. As used herein, the term "complementary sequences" means
nucleic acid sequences which are substantially complementary, as
may be assessed by the same nucleotide comparison set forth above,
or as defined as being capable of hybridizing to the nucleic acid
segment in question under relatively stringent conditions such as
those described herein.
[0067] Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, or organic solvents,
in addition to the base composition, length of the complementary
strands, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. Stringent temperature conditions will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and preferably in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1,000 mM,
typically less than 500 mM, and preferably less than 200 mM.
However, the combination of parameters is much more important than
the measure of any single parameter. See e.g., Wetmur &
Davidson, (1968).
[0068] Probe sequences may also hybridize specifically to duplex
DNA under certain conditions to form triplex or other higher order
DNA complexes. The preparation of such probes and suitable
hybridization conditions are well known in the art.
[0069] As used herein, the term "DNA segment" refers to a DNA
molecule which has been isolated free of total genomic DNA of a
particular species. Furthermore, a DNA segment encoding a GB1
domain polypeptide refers to a DNA segment which contains GB1
domain polypeptide coding sequences, yet is isolated away from, or
purified free from, total genomic DNA of a source species, such as
Staphyloccocus sp. or Streptoccocus sp. Included within the term
"DNA segment" are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phages, viruses, and the like.
[0070] Similarly, a DNA segment comprising an isolated or purified
GB1 domain-encoding nucleic acid refers to a DNA segment including
GB1 domain coding sequences isolated substantially away from other
naturally occurring polypeptide or protein encoding sequences. In
this respect, the term "GB1 domain-encoding nucleic acid" is used
for simplicity to refer to a functional protein, polypeptide or
peptide encoding unit. "Isolated substantially away from other
coding sequences" means that the nucleic acid of interest, in this
case, the GB1 domain-encoding nucleic acid, forms the significant
part of the coding region of the DNA segment, and that the DNA
segment does not contain large portions of naturally-occurring
coding DNA, such as large chromosomal fragments or other functional
nucleic acids or coding regions. Of course, this refers to the DNA
segment as originally isolated, and does not exclude GB1
domain-encoding nucleic acids or coding regions later added to the
segment by the hand of man.
[0071] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
which encode a GB1 domain polypeptide that includes within its
amino acid sequence an amino acid sequence selected from any of SEQ
ID NO's:6, 8, 10, 12, 16, 18, 20, 22 and 24. It will also be
understood that this invention is not limited to the particular
nucleic acid and amino acid sequences of SEQ ID NO's: 5-12 and
15-24. Recombinant vectors and isolated DNA segments may therefore
variously include the GB1 domain polypeptide-encoding region
itself, include coding regions bearing selected alterations or
modifications in the basic coding region, or include encoded larger
polypeptides which nevertheless include GB1 domain
polypeptide-encoding regions or may encode biologically functional
equivalent proteins or peptides which have variant amino acid
sequences.
[0072] In certain embodiments, the invention concerns isolated DNA
segments and recombinant vectors which encode a protein or peptide
that includes within its amino acid sequence an amino acid sequence
essentially as set forth in any of SEQ ID NO's:6, 8, 10, 12, 16,
18, 20, 22 and 24. Naturally, where the DNA segment or vector
encodes a full length GB1 domain-encoding nucleic acid product, a
most preferred nucleic acid sequence comprises any of those which
are essentially as set forth in SEQ ID NO's: 5, 7, 9, 11, 15, 17,
19, 21 and 23, and which encode a polypeptide which binds an IgG
Fab fragment but substantially does not bind an IgG Fc
fragment.
[0073] The term "a sequence essentially as set forth in any of SEQ
ID NO's:6, 8, 10, 12, 16, 18, 20, 22 and 24" means that the
sequence substantially corresponds to a portion an amino acid
sequence of any of SEQ ID NO's:6, 8, 10, 12, 16, 18, 20, 22 and 24
and has relatively few amino acids which are not identical to, or a
biologically functional equivalent of, the amino acids of an amino
acid sequence of any of SEQ ID NO's:6, 8, 10, 12, 16, 18, 20, 22
and 24. The term "biologically functional equivalent" is well
understood in the art and is further defined in detail herein.
Accordingly, sequences, which have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more
preferably, between about 91% and about 99%; of amino acids which
are identical or functionally equivalent to the amino acids of SEQ
ID NO's:6, 8, 10, 12, 16, 18, 20, 22 and 24, will be sequences
which comprise "a sequence essentially as set forth in any of SEQ
ID NO's:6, 8, 10, 12, 16, 18, 20, 22 and 24".
[0074] In certain other embodiments, the invention concerns
isolated DNA segments and recombinant vectors that include within
their sequence a nucleic acid sequence essentially as set forth in
any of SEQ ID NO's: 5, 7, 9, 11, 15, 17, 19, 21 and 23. The term "a
sequence essentially as set forth in any of SEQ ID NO's: 5, 7, 9,
11, 15, 17, 19, 21 and 23" is used in the same sense as described
above and means that the nucleic acid sequence substantially
corresponds to a portion of any of SEQ ID NO's: 5, 7, 9, 11, 15,
17, 19, 21 and 23, respectively, and has relatively few codons
which are not identical, or functionally equivalent, to the codons
of any of SEQ ID NO's: 5, 7, 9, 11, 15, 17, 19, 21 and 23,
respectively. Again, DNA segments which encode polypeptides
exhibiting Fab binding activity but substantially no Fc binding
activity will be most preferred. The term "functionally equivalent
codon" is used herein to refer to codons that encode the same amino
acid, such as the six codons for arginine or serine, and also to
refer to codons that encode biologically equivalent amino acids
(see Table 2).
[0075] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, may be
combined with other DNA sequences, such as promoters, enhancers,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments may be prepared which
include a short stretch complementary to a nucleic acid sequence
set forth in any of SEQ ID NO's: 5, 7, 9, 11, 15, 17, 19, 21 and
23, respectively, such as about 10 nucleotides, for use as a PCR
primer, for example. Alternatively, longer nucleic acid molecules
may be prepared which are up to 1,000 or 500 base pairs in length,
with segments of 200 being preferred in certain cases. DNA segments
with total lengths of about 250, 200, 150, 100 and about 50 base
pairs in length are also contemplated to be useful.
[0076] The DNA segments of the present invention encompass
biologically functional equivalent GB1 domain proteins and
polypeptides. Such sequences may rise as a consequence of codon
redundancy and functional equivalency which are known to occur
naturally within nucleic acid sequences and the proteins thus
encoded. Alternatively, functionally equivalent proteins or
peptides may be created via the application of recombinant DNA
technology, in which changes in the protein structure may be
engineered, based on considerations of the properties of the amino
acids being exchanged. Changes designed by man may be introduced
through the application of site-directed mutagenesis techniques,
e.g., to introduce changes in IgG Fab and IgG Fc binding activity
or to test GB1 domain mutants in orderto examine IgG Fab and IgG Fc
binding activity at the molecular level.
[0077] If desired, one may also prepare fusion proteins and
peptides, e.g., where the GB1 domain coding region is aligned
within the same expression unit with other proteins or peptides
having desired functions, such as for purification or
immunodetection purposes (e.g., proteins which may be purified by
affinity chromatography and enzyme label coding regions,
respectively).
[0078] Recombinant vectors form important further aspects of the
present invention. Particularly useful vectors are contemplated to
be those vectors in which the coding portion of the DNA segment is
positioned under the control of a promoter. The promoter may be in
the form of the promoter which is naturally associated with the GB1
domain-encoding nucleic acid, e.g., in bacterial cells, as may be
obtained by isolating the 5' non-coding sequences located upstream
of the coding segment, for example, using recombinant cloning
and/or PCR technology, in connection with the compositions
disclosed herein.
[0079] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with a GB1
domain-encoding nucleic acid in its natural environment. Such
promoters may include promoters isolated from bacterial, viral,
eukaryotic, or mammalian cells. Naturally, it will be important to
employ a promoter that effectively directs the expression of the
DNA segment in the cell type chosen for expression. The use of
promoter and cell type combinations for protein expression is
generally known to those of skill in the art of molecular biology,
for example, see Sambrook et al., (1989) Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.) and Ausubel et al. (1992) Current Protocols in
Molecular Biology, (J. Wylie & Sons, New York, N.Y.),
specifically incorporated herein by reference. The promoters
employed may be constitutive, or inducible, and can be used under
the appropriate conditions to direct high level expression of the
introduced DNA segment, such as is advantageous in the large-scale
production of recombinant proteins or peptides. Appropriate
promoter systems contemplated for use in high-level expression
include, but are not limited to, the vaccina virus promoter and the
baculovirus promoter.
[0080] In an alternative embodiment, the present invention provides
an expression vector comprising a polynucleotide that encodes a GB1
domain polypeptide exhibiting Fab binding activity but
substantially no Fc binding activity, or other binding activity in
accordance with the present invention. More preferably, an
expression vector of the present invention comprises a
polynucleotide that encodes a polypeptide comprising an amino acid
residue sequence of any of SEQ ID NO's:6, 8, 10, 12, 16, 18, 20, 22
and 24, respectively. More preferably, an expression vector of the
present invention comprises a polynucleotide comprising the
nucleotide base sequence of any of SEQ ID NO's: 5, 7, 9, 11, 15,
17, 19, 21 and 23, respectively.
[0081] Even more preferably, an expression vector of the invention
comprises a polynucleotide operatively linked to an
enhancer-promoter. More preferably still, an expression vector of
the invention comprises a polynucleotide operatively linked to a
prokaryotic promoter. Alternatively, an expression vector of the
present invention comprises a polynucleotide operatively linked to
an enhancer-promoter that is a eukaryotic promoter, and the
expression vector further comprises a polyadenylation signal that
is positioned 3' of the carboxy-terminal amino acid and within a
transcriptional unit of the encoded polypeptide.
[0082] In yet another embodiment, the present invention provides a
recombinant host cell transfected with a polynucleotide that
encodes a GB1 domain polypeptide a GB1 domain polypeptide
exhibiting Fab binding activity but substantially no Fc binding
activity, or other binding activity in accordance with the present
invention. SEQ ID NO's:5-12 and 15-24 set forth exemplary
nucleotide and amino acid sequences. A host cell of the invention
may comprise a eukaryotic host cell, such as a vertebrate cell, or
more particularly, a mammalian cell.
[0083] In another aspect, a recombinant host cell of the present
invention is a prokaryotic host cell. Preferably, a recombinant
host cell of the invention is a bacterial cell, preferably a strain
of Escherichia coli (E. coli). More preferably, a recombinant host
cell comprises a polynucleotide under the transcriptional control
of regulatory signals functional in the recombinant host cell,
wherein the regulatory signals appropriately control expression of
the GB1 domain polypeptide in a manner to enable all necessary
transcriptional and post-transcriptional modification.
[0084] In yet another embodiment, the present invention
contemplates a process of preparing a GB1 domain polypeptide
comprising transfecting a cell with polynucleotide that encodes a
GB1 domain polypeptide exhibiting Fab binding activity but
substantially no Fc binding activity, or other binding activity in
accordance with the present invention, to produce a transformed
host cell; and maintaining the transformed host cell under
biological conditions sufficient for expression of the polypeptide.
More preferably, the transformed host cell is a eukaryotic cell.
More preferably still, the eukaryotic cell is a vertebrate cell.
Alternatively, the host cell is a prokaryotic cell. More
preferably, the prokaryotic cell is a bacterial cell of Escherichia
coli. Even more preferably, a polynucleotide transfected into the
transformed cell comprises the nucleotide base sequence of any of
SEQ ID NO's: 5, 7, 9, 11, 15, 17, 19, 21 and 23, respectively.
[0085] Any suitable known method of protein purification may be
used to recover and purify the GB1 domain polypeptide from the host
cells. The cells may be lysed, if necessary, using known chemical,
physical, and/or enzymatic means. The polypeptides then may be
purified from the cell lysate using such standard procedures as
adsorption to immobilized immunoglobulin, as described by Sioquist,
U.S. Pat. No. 3,850,798 (1974), ion-exchange or gel chromatography,
precipitation (e.g., with ammonium sulfate), dialysis, filtration,
or a combination of these methods.
[0086] As mentioned above, in connection with expression
embodiments to prepare recombinant GB1 domain proteins and
peptides, it is contemplated that longer DNA segments will most
often be used, with DNA segments encoding the entire GB1 domain
polypeptide being most preferred. However, it will be appreciated
that the use of shorter DNA segments to direct the expression of
GB1 domain peptides, Fab binding regions or Fc binding regions,
such as may be used to test binding characteristics, also falls
within the scope of the invention.
[0087] DNA segments which encode GB1 domain polypeptide segments
from about 15 to about 45 amino acids in length, or more
preferably, from about 10 to about 20 amino acids in length are
contemplated to be particularly useful. DNA segments encoding
peptides will generally have a minimum coding length in the order
of about 45 to about 150, or to about 30 to about 60 nucleotides.
DNA segments encoding full length proteins may have a minimum
coding length on the order of about 200 nucleotides for a
polypeptide essentially as set forth in any of SEQ ID NO's:6, 8,
10, 12, 16, 18, 20, 22 and 24, respectively.
[0088] Naturally, the present invention also encompasses DNA
segments which are complementary, or essentially complementary, to
the sequences set forth in any of SEQ ID NO's: 5, 7, 9, 11, 15, 17,
19, 21 and 23, respectively. The terms "complementary" and
"essentially complementary" are defined above. Excepting intronic
or flanking regions, and allowing for the degeneracy of the genetic
code, sequences which have between about 70% and about 80%; or more
preferably, between about 81% and about 90%; or even more
preferably, between about 91% and about 99%; of nucleotides which
are identical or functionally equivalent (i.e. encoding the same
amino acid) of nucleotides of any of SEQ ID NO's: 5, 7, 9, 11, 15,
17, 19, 21 and 23, respectively, will be sequences which are, "a
sequence essentially as set forth in any of SEQ ID NO's: 5, 7, 9,
11, 15, 17, 19, 21 and 23, respectively". Sequences which are
essentially the same as those set forth in any of SEQ ID NO's: 5,
7, 9, 11, 15, 17, 19, 21 and 23, respectively, may also be
functionally defined as sequences which are capable of hybridizing
to a nucleic acid segment containing the complement of any of SEQ
ID NO's: 5, 7, 9, 11, 15, 17, 19, 21 and 23, respectively, under
relatively stringent conditions. Suitable relatively stringent
hybridization conditions are described herein and are well known to
those of skill in the art.
[0089] E. Biologically Functional Equivalents
[0090] As mentioned above, modification and changes may be made in
the structure of the GB1 peptides described herein and still obtain
a molecule having like or otherwise desirable characteristics. For
example, certain amino acids may be substituted for other amino
acids in a protein structure without appreciable loss of
interactive capacity with an Fab fragment of IgG. Additionally,
certain amino acids may be substituted for other amino acids in a
protein structure without appreciable loss of disrupted interactive
capacity with an Fc fragment of an IgG.
[0091] Since it is the interactive capacity and nature of a protein
that defines that protein's biological activity, certain amino acid
sequence substitutions can be made in a protein sequence (or, of
course, its underlying DNA coding sequence) and nevertheless obtain
a protein with like or even countervailing properties (e.g.,
binding v. substantially not binding). It is thus contemplated by
the inventors that various changes may be made in the sequence of
the GB1 domain polypeptides (or underlying DNA) without appreciable
loss of their IgG Fab binding utility or their disrupted binding
capacity with an Fc fragment of an IgG.
[0092] It is also well understood by the skilled artisan that,
inherent in the definition of a biologically functional equivalent
protein or peptide, is the concept that there is a limit to the
number of changes that may be made within a defined portion of the
molecule and still result in a molecule with an acceptable level of
equivalent binding or other biological activity. Biologically
functional equivalent peptides are thus defined herein as those
peptides in which certain, not most or all, of the amino acids may
be substituted. Of course, a plurality of distinct
proteins/peptides with different substitutions may easily be made
and used in accordance with the invention.
[0093] It is also well understood that where certain residues are
shown to be particularly important to the biological or structural
properties of a protein or peptide, e.g., residues in binding or
other active sites, such residues may not generally be exchanged.
This is the case in the present invention, where any changes, for
example, in the Fab-binding site characterized by backbone contacts
between the edge of the .beta.-sheet of the B1 domain polypeptide
and the last .beta.-strand of the C.sub.H1 domain of the Fab
fragment, could result in a loss of an aspect of the Fab binding
utility of the resulting peptide for the present invention.
[0094] Amino acid substitutions, such as those which might be
employed in modifying the GB1 domain polypeptides described herein,
are generally based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. An analysis of the
size, shape and type of the amino acid side-chain substituents
reveals that arginine, lysine and histidine are all positively
charged residues; that alanine, glycine and serine are all a
similar size; and that phenylalanine, tryptophan and tyrosine all
have a generally similar shape. Therefore, based upon these
considerations, arginine, lysine and histidine; alanine, glycine
and serine; and phenylalanine, tryptophan and tyrosine; are defined
herein as biologically functional equivalents.
[0095] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(4.5). The amino acids having a positive hydropathic index may also
be referred to as "non-polar" residues, while the amino acids
having a negative hydropathic index may also be referred to as
"polar" residues.
[0096] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte & Doolittle (1982),
incorporated herein by reference). It is known that certain amino
acids may be substituted for other amino acids having a similar
hydropathic index or score and still retain a similar biological
activity. In making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indices are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred.
[0097] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.
with a biological property of the protein. It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein.
[0098] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phehylalanine (-2.5); tryptophan (-3.4). The amino acids having a
positive hydrophilic index may also be referred to as "polar"
residues, while the amino acids having a negative hydrophilic index
may also be referred to as "non-polar" residues, or as
"comparatively non-polar" residues when compared to amino acids
having a positive hydrophilic index.
[0099] In making changes based upon similar hydrophilicity values,
the substitution of amino acids whose hydrophilicity values are
within .+-.2 is preferred, those which are within .+-.1 are
particularly preferred, and those within 0.5 are even more
particularly preferred.
[0100] While discussion has focused on functionally equivalent
polypeptides arising from amino acid changes, it will be
appreciated that these changes may be effected by alteration of the
encoding DNA, taking into consideration also that the genetic code
is degenerate and that two or more codons may code for the same
amino acid.
[0101] F. Sequence Modification Techniques
[0102] Modifications to the GB1 domain polypeptides described
herein may be carried out using techniques such as site directed
mutagenesis. Site-specific mutagenesis is a technique useful in the
preparation of individual peptides, or biologically functional
equivalent proteins or peptides, through specific mutagenesis of
the underlying DNA. The technique further provides a ready ability
to prepare and test sequence variants, for example, incorporating
one or more of the foregoing considerations, by introducing one or
more nucleotide sequence changes into the DNA. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 30 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0103] In general, the technique of site-specific mutagenesis is
well known in the art as exemplified by publications (e.g., Adelman
et al. (1983)). As will be appreciated, the technique typically
employs a phage vector which exists in both a single stranded and
double stranded form. Typical vectors useful in site-directed
mutagenesis include vectors such as the M13 phage (Messing et al.,
1981). These phage are readily commercially available and their use
is generally well known to those skilled in the art. Double
stranded plasmids are also routinely employed in site directed
mutagenesis which eliminates the step of transferring the gene of
interest from a plasmid to a phage. A polymerase chain reaction
(PCR) based site-directed mutagenesis technique is disclosed in the
Examples.
[0104] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart the two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes, for example, the
B1 domain of protein G. An oligonucleotide primer bearing the
desired mutated sequence is prepared, generally synthetically, for
example by the method of Crea et al. (1978). This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement.
[0105] G. Peptide Synthesis Techniques
[0106] Alternatively, the GB1 domain polypeptides of the present
invention may be prepared by a peptide synthesis techniques. Such
techniques are contemplated for use in preparing a polypeptide of
the present invention which comprises no more than about 100 amino
acid residues, preferably no more than about 80 residues, and more
preferably no more than about 60 residues. Synthesized peptides can
be linear or cyclic.
[0107] As used herein and in the claims, a "GB1 domain polypeptide"
of the present invention includes any analog, fragment or chemical
derivative of a GB1 domain polypeptide having desired binding
characteristics. Such a polypeptide can be subject to various
changes, substitutions, insertions, and deletions where such
changes provide for certain advantages in its use. In this regard,
an GB1 domain polypeptide for use in a purification method of the
present invention corresponds to, rather than is identical to, the
sequence of a native GB1 domain polypeptide, where one or more
changes are made and it retains binding activity for IgG Fab
fragments and substantially no binding activity for IgG Fc
fragments as disclosed herein. Thus, a polypeptide can be in any of
a variety of forms of peptide derivatives, that include amides,
conjugates with proteins, cyclized peptides, polymerized peptides,
analogs, fragments, chemically modified peptides, and the like
derivatives.
[0108] The term "analog" includes any polypeptide having an amino
acid residue sequence substantially identical to a sequence of a
naturally occurring GB1 domain polypeptide in which one or more
residues have been conservatively substituted with a functionally
similar residue and which displays binding activity for IgG Fab
fragments and substantially no binding activity for IgG Fc
fragments as described herein. Examples of conservative
substitutions include the substitution of one non-polar
(hydrophobic) residue such as alanine, isoleucine, valine, leucine
or methionine for another; the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine; the substitution of one basic residue such as lysine,
arginine or histidine for another; or the substitution of one
acidic residue, such as aspartic acid or glutamic acid for another.
Such substitutions are described in detail above with respect to an
isolated and purified GB1 domain polypeptide of the present
invention.
[0109] The phrase "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue provided that such polypeptide displays the requisite
binding activity. "Chemical derivative" refers to a subject
polypeptide having one or more residues chemically derivatized by
reaction of a functional side group. Such derivatized molecules
include for example, those molecules in which free amino groups
have been derivatized to form amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-im-benzylhistidine. Also
included as chemical derivatives are those peptides which contain
one or more naturally occurring amino acid derivatives of the
twenty standard amino acids. For examples: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be
substituted for lysine. Polypeptides of the present invention also
include any polypeptide having one or more additions and/or
deletions or residues relative to the sequence of a polypeptide
whose sequence is shown herein, so long as the requisite binding
activity is maintained.
[0110] The term "fragment" refers to any subject polypeptide having
an amino acid residue sequence shorter than that of a polypeptide
whose amino acid residue sequence is shown herein.
[0111] When a polypeptide of the present invention has a sequence
that is not identical to the sequence of a native GB1 domain
polypeptide, it is typically because one or more conservative or
non-conservative substitutions have been made, usually no more than
about 30 number percent, and preferably no more than 10 number
percent of the amino acid residues are substituted. Additional
residues may also be added at either terminus of a polypeptide for
the purpose of providing a "linker" by which the polypeptides of
this invention can be conveniently affixed to a label or solid
matrix, or carrier. Labels, solid matrices and carriers that can be
used with the polypeptides of this invention are described
elsewhere herein.
[0112] Amino acid residue linkers are usually at least one residue
and can be 40 or more residues, more often 1 to 10 residues, but do
not form GB1 domain polypeptide epitopes. Typical amino acid
residues used for linking are tyrosine, cysteine, lysine, glutamic
and aspartic acid, or the like. In addition, a subject polypeptide
can differ, unless otherwise specified, from the natural sequence
of a GB1 domain polypeptide by the sequence being modified
byterminal-NH2 acylation, e.g., acetylation, or thioglycolic acid
amidation, by terminal-carboxylamidation, e.g., with ammonia,
methylamine, and the like terminal modifications. Terminal
modifications are useful, as is well known, to reduce
susceptibility by proteinase digestion, and therefore serve to
prolong half life of the polypeptides in solutions, particularly
biological fluids where proteases may be present. In this regard,
polypeptide cyclization is also a useful terminal modification, and
is particularly preferred also because of the stable structures
formed by cyclization and in view of the biological activities
observed for such cyclic peptides as described herein.
[0113] Any peptide of the present invention may be used in the form
of a pharmaceutically acceptable salt. Suitable acids which are
capable of the peptides with the peptides of the present invention
include inorganic acids such as trifluoroacetic acid (TFA),
hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric
acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic
acid, malonic acid, succinic acid, maleic acid, fumaric, acid,
anthranilic acid, cinnamic acid, naphthalene sulfonic acid,
sulfanilic acid or the like. HCl and TFA salts are particularly
preferred.
[0114] Suitable bases capable of forming salts with the peptides of
the present invention include inorganic bases such as sodium
hydroxide, ammonium hydroxide, potassium hydroxide and the like;
and organic bases such as mono- di- and tri-alkyl and aryl amines
(e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl
amine and the like), and optionally substituted ethanolamines (e.g.
ethanolamine, diethanolamine and the like).
[0115] A peptide of the present invention, also referred to herein
as a subject polypeptide, can be synthesized by any of the
techniques that are known to those skilled in the polypeptide art,
including recombinant DNA techniques. Synthetic chemistry
techniques, such as a solid-phase Merrifield-type synthesis, are
preferred for reasons of purity, antigenic specificity, freedom
from undesired side products, ease of production and the like. An
excellent summary of the many techniques available can be found in
Steward et al. (1969) "Solid Phase Peptide Synthesis", W. H.
Freeman Co., San Francisco, Calif.; Bodanszkv et al. (1976)
"Peptide Synthesis", John Wiley & Sons, Second Edition;
Meienhofer, J. (1983) "Hormonal Proteins and Peptides", Vol. 2, p.
46, Academic Press, New York, N.Y.; Merrifield (1969) Adv. Enzymol.
32:221-96; Fields et al. (1990) Int. J. Peptide Protein Res.,
35:161-214; U.S. Pat. No. 4,244,946 for solid phase peptide
synthesis, and Schroder et al. (1965) "The Peptides", Vol. 1,
Academic Press, New York, N.Y. for classical solution synthesis,
each of which is incorporated herein by reference. Appropriate
protective groups usable in such synthesis are described in the
above texts and in McOmie. J. F. W. (1973) "Protective Groups in
Organic Chemistry", Plenum Press, New York, N.Y., which is
incorporated herein by reference.
[0116] In general, the solid-phase synthesis methods contemplated
comprise the sequential addition of one or more amino acid residues
or suitably protected amino acid residues to a growing peptide
chain. Normally, either the amino or carboxyl group of the first
amino acid residue is protected by a suitable, selectively
removable protecting group. a different, selectively removable
protecting group is utilized for amino acids containing a reactive
side group such as lysine.
[0117] Using a solid phase synthesis as exemplary, the protected or
derivatized amino acid is attached to an inert solid support
through its unprotected carboxyl or amino group. The protecting
group of the amino or carboxyl group is then selectively removed
and the next amino acid in the sequence having the complimentary
(amino or carboxyl) group suitably protected is admixed and reacted
under conditions suitable for forming the amide linkage with the
residue already attached to the solid support. The protecting group
of the amino or carboxyl group is then removed from this newly
added amino acid residue, and the next amino acid (suitably
protected) is then added, and so forth. After all the desired amino
acids have been linked in the proper sequence, any remaining
terminal and side group protecting groups (and solid support) are
removed sequentially or concurrently, to afford the final linear
polypeptide.
[0118] The resultant linear polypeptides prepared for example as
described above may be reacted to form their corresponding cyclic
peptides. An exemplary method for cyclizing peptides is described
by Zimmer et al. (1993) Peptides 1992, pp. 393-394, ESCOM Science
Publishers, B. V. Typically, tertbutoxycarbonyl protected peptide
methyl ester is dissolved in methanol and sodium hydroxide solution
are added and the admixture is reacted at 20.degree. C. to
hydrolytically remove the methyl ester protecting group. After
evaporating the solvent, the tertbutoxycarbonyl protected peptide
is extracted with ethyl acetate from acidified aqueous solvent. The
tertbutoxycarbonyl protecting group is then removed under mildly
acidic conditions in dioxane cosolvent. The unprotected linear
peptide with free amino and carboxy termini so obtained is
converted to its corresponding cyclic peptide by reacting a dilute
solution of the linear peptide, in a mixture of dichloromethane and
dimethylformamide, with dicyclohexylcarbodiimide in the presence of
1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclic
peptide is then purified by chromatography.
[0119] H. Detection and Precipitation Methods
[0120] Binding substances comprising the GB1 polypeptides of the
present invention have selective binding activity with an IgG, a
fragment of an IgG, or combinations thereof, independent of the
antibody epitope (antigen recognition specificity). Such binding
does not occur between the binding substance comprising the GB1
polypeptides of the present invention and other serum components.
Thus, this binding specificity can be employed for detecting and/or
purifying an IgG, a fragment of an IgG, or combinations thereof.
The term "fragment" thus refers any fragment of an IgG, including
fragments that bind the GB1 polypeptides of the present invention,
such as Fab and F(ab').sub.2 fragments.
[0121] GB1 polypeptides of the present invention are prepared as
described herein above. The polypeptide is then conjugated to, or
labeled with, a material that will enable visualization of the
presence of the GB1 polypeptide.
[0122] The GB1 domain polypeptides of the present invention can
thus be used in a variety of applications to detect antibodies or
antibody fragments. For example, fluoresceinated, alkaline
phosphatase labeled, peroxidase labeled, or biotinylated GB1 domain
polypeptides of the present invention are used in indirect
cytochemical assays to detect antibody binding to cells and tissues
in histological or flow cytometric assays. Particularly, the GB1
polypeptides of the present invention labeled in this manner detect
the Fab portion of an antibody molecule, and such detection may be
used in a variety of research or clinical contexts.
[0123] Similarly, immobilized GB1 polypeptides of the present
invention can be used to precipitate immune complexes in
radioimmune and other quantitative immune or antigen capture
assays. Such immunoprecipitation assays where immune complexes of
radiolabeled antigens are captured on immobilized GB1 polypeptides
of the present invention have wide application in the art.
[0124] By way of elaboration, the GB1 polypeptides of the present
invention are used to detect the presence of IgG antibodies
fragments thereof, in solutions, or on surfaces exposed to IgG
antibodies, or fragments thereof, by a variety of techniques.
Techniques which are used include: enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), immunoblot analysis,
immunofluorescent assay (IFA), immunohistochemistry, immunoelectron
microscopy (IEM), and immunoilluminescence. Each technique utilizes
conjugates including the GB1 polypeptides of the present invention
to visualize the binding of the conjugate to IgG antibody molecules
or fragments thereof.
[0125] Commonly used conjugates include, but are not limited to,
enzymes such as biotin, horseradish peroxidase, alkaline
phosphatase (O'Sullivan et al. (1978) FEBS Letters 95:311), acid
phosphatase, beta-galactosidase (Ishikawa et al. (1978) Scand. J.
Immunol. 8:43) and luciferase; radioisotopes such as .sup.125I,
.sup.35S, .sup.14C, and .sup.3H; fluorescent dyes such as
fluorescein, rhodamine, dichlorotriazinylaminofl- uorescein (DTAF;
Blakeslee et al., J. Immunol. Meth. 13:320 (1977)), ferritin
(Carlsson et al. (1978) Biochem. J. 173:723), fluoroscene
isothiocyanste (FITC; McKinney et al. (1966) Anal. Biochem.
14:421), sulforhodamine 101 acid chloride (Texas Red) and
tetra-methyrhodamine isothiocyanate (TRITC; Amante et al., J.
Immunol. Meth., 1:289 (1972)); colloidal gold particles
(Horisberger et al., Histochem. 82:219 (1985)); and the like.
Effective procedures for such conjugations are generally
conventional, as described by Harlow et al., 1988, Antibodies: a
laboratory manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
[0126] The protein conjugate is stored in appropriate buffers until
needed. Colloidal gold conjugates may be maintained in Tris-based
stabilizing buffer, such as those described in Robinson et al.,
(1984) Infect. Immun. 46:361-366. For other conjugates, the buffer
would typically be phosphate-buffered saline, pH 7.2 (PBS).
However, physiological buffers such as Tris- or borate-buffered
saline (TBS or BBS) in pH ranging from 6.5 to 8.0, or non-saline
buffers such as acetates, bicarbonates, or citrates within this pH
range may be utilized.
[0127] When needed to detect the presence of IgG antibodies or
fragments thereof in a preparation, the GB1 polypeptide conjugate
may be first diluted in an appropriate buffer. The extent of
dilution varies according to the conjugate and sensitivity
required, and is normally determined empirically for a given
conjugate preparation and detection method. Dilutions typically
range from 1:10 to 1:10,000. After dilution the conjugate is
incubated with a sample suspected of containing IgG antibodies or
fragments thereof. The incubation should proceed for about 15-60
minutes at room temperature, or about 4-16 hours at about 4.degree.
C., during which time from one to ten (optimally) GB1 polypeptide
molecules will bind to any IgG antibodies or fragments thereof
present. Following incubation, the sample is washed twice for about
5-10 minutes each with dilution buffer or with buffer which is
compatible with the visualization conditions (if different). The
presence of bound GB1 polypeptide may then be detected or
visualized by chromogenic assay, radioactivity, illuminescence,
fluorescence, flow cytometry or electron density, as appropriate
for the conjugate.
[0128] Thus, a method for detecting IgG, a fragment of an IgG, or
combinations thereof, in a sample suspected of containing IgG, a
fragment of an IgG, or combinations thereof, is provided in
accordance with the present invention. The method comprising the
steps of: (a) contacting the sample with a binding substance
comprising the GB1 polypeptide of the present invention under
conditions favorable to binding of IgG, a fragment of an IgG, or
combinations thereof, to the binding substance to form a complex
therebetween; and (b) detecting the complex by means of a label
conjugated to the binding substance or by means of a labeled
reagent that specifically binds to the complex subsequent to its
formation.
[0129] In the detection method of the present invention, the
binding substance can be immobilized on a solid substrate. In such
case, the detecting step (b) comprises: (i) contacting the complex
with a reagent conjugated with a detectable label wherein the
reagent specifically binds to IgG, a fragment of an IgG, or
combinations thereof, and (ii) detecting the detectable label.
[0130] In the detection method of the present invention, the
binding substance can be conjugated with a detectable label. In
such case, the detecting step (b) comprises: (i) separating the
complex from unbound labeled binding substance; and (ii) detecting
the detectable label which is present in the complex or which is
unbound.
[0131] The detection method of the present invention can further
comprise: (i) contacting the complex with a reagent immobilized on
a solid substrate to form immobilized complex thereon wherein the
reagent binds to IgG, a fragment of an IgG, or combinations
thereof, present in the complexes; and (ii) separating the
immobilized complex from the remaining mixture.
[0132] I. Purification of IgG's and IgG Fragments
[0133] The immobilized GB1 domain polypeptides of the present
invention may be used to separate or bind IgG's or fragments
thereof. In particular, the immobilized GB1 domain polypeptides of
the present invention bind Fab and F(ab').sub.2 fragments of IgG's
and substantially do not bind Fc fragments of IgG's. Therefore, the
immobilized GB1 polypeptides are particularly useful for the
separation of IgG Fc fragments from IgG Fab fragments.
[0134] Conjugates of GB1 proteins may thus used to purify IgG's, a
fragment of an IgG, or combinations thereof by methods similar to
those used for purification of IgG antibodies with protein A. Such
affinity purification methods generally utilize insoluble or
immobile protein conjugates to facilitate eventual separation of
the antibody and the immunoglobulin-binding protein and to separate
antibody fragments. A purification procedure for IgG, a fragment of
an IgG, or combinations thereof, uses a GB1 polypeptide of the
present invention conjugated to an insoluble matrix by methods such
as those described previously by Harlow et al. (1988) Antibodies: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. Appropriate matrices may include but are not limited
to agarose, latex, magnetic or polyacrylamide beads, silica or
polystyrene.
[0135] Following conjugation, any available sites on the matrix are
blocked by appropriate reagents, and the conjugate can be used
immediately or prepared for storage. The conjugates are preferably
stored under conditions offering the greatest stability. Although,
such conditions vary for different conjugates, optimum stability is
usually achieved by dehydration, and preferably cooling to about
-20.degree. C. or less. Conjugates can also be stored hydrated in
physiological buffers, at about 4.degree. C., with the addition of
an antimicrobial preservative. Suitable buffers include but are not
limited to PBS, TBS, BBS, or non-saline buffers which prove
effective. Typically, merthiolate (thimerosol) or sodium azide is
added at 0.01 to 0.05% to retard microbial growth. Azides should
not be used when conjugates are stored in metal containers.
[0136] The conjugate is then loaded into a vessel to be used for
incubation with a source of IgG, fragments of an IgG, or
combinations thereof. Chromatography columns are the preferred
incubation vessel. These can be constructed from glass, plastics,
or metals, depending on the desired volume and pressure
constraints. A wide range of chromatography columns are
commercially available for this purpose. The volume of conjugate
loaded into the vessel varies according to the dimensions of the
vessel, and the quantity of IgG, fragments of an IgG, or
combinations thereof, expected to be purified. Optimally, this
volume corresponds to the binding capacity of the GB1 polypeptide
conjugate, which is determined experimentally for each conjugate
preparation. A value of about 1-20 mg per ml of conjugate is
generally preferred.
[0137] After loading into a vessel, the conjugate may be
equilibrated with a buffer compatible with the interaction between
the GB1 polypeptide conjugate and an IgG, a fragment of an IgG, or
combinations thereof interaction. Suitable buffers include those
described above for conjugate storage, except that no preservative
would be added. Once equilibrated, a sample containing IgG, a
fragment of an IgG, or combinations thereof, is added to the
vessel. Optimally, a volume of sample sufficient to completely wet
the conjugate is added. This usually represents about 0.3-0.5
volumes of conjugate. Optionally, the sample containing IgG,
fragments of an IgG, or combinations thereof is mammalian serum. If
not, the sample is preferably a solution in the buffer used to
equilibrate the column.
[0138] The column and the sample are incubated at room temperature
for about 15-60 minutes, then washed thoroughly with equilibration
buffer. Buffer passing through the column is monitored for
macromolecule content until such content is negligible. This is
usually accomplished by measuring the absorbance (280 nm) of the
buffer until it returns to the baseline value of the buffer. Buffer
fractions can be collected, in that in accordance with one
embodiment of the present invention, Fc fragments which are
substantially not bound by the GB1 polypeptide are isolated.
[0139] After washing, IgG's, fragments of an IgG, or combinations
thereof, retained within the matrix are eluted by the addition of
an agent which dissociates the IgG's, fragment of an IgG, or
combinations thereof from the GB1 polypeptide conjugate. Examples
of two of these agents are sodium dodecylsulfate (SDS) ranging in
concentration from 0.1-2%, and CCS buffer comprised of 0.02M sodium
cacodylate, 0.010M calcium carbonate, and 0.2M sucrose, pH 7.2,
containing 0.1-1 unit of neuraminidase.
[0140] Up to 90% or greater of the IgG, fragments of an IgG, or
combinations thereof is dissociated with SDS. Further dissociation
of minute quantities of IgG, fragments of an IgG, or combinations
thereof are observed with the latter treatment.
[0141] Additional reagents suitable for the elution of IgG's or
fragments thereof antibodies may also be employed. These include
but are not limited to agents which alter the pH, salt
concentration, or hydrophobic interactions affecting the GB1
interaction with IgG's or fragments thereof.
[0142] Once eluted, the IgG, fragments of an IgG, or combinations
thereof are dialysed thoroughly against a volatile aqueous solvent,
which may include solutions of 0.010 to 0.2M ammonium bicarbonate,
or ammonium acetate, or distilled water, and then dried in a vacuum
evaporator. The dried antibodies is preferably desiccated and
stored at about 4.degree. C. Freezing of the IgG, fragments of an
IgG, or combinations thereof antibodies should be avoided.
Alternatively, the IgG, fragments of an IgG, or combinations
thereof can be dialysed into a suitable physiological buffer (see
above), concentrated in a vacuum concentrator, and stored in the
buffer, supplemented with an antimicrobial agent, as described
above.
[0143] In accordance with the present invention, a method for
purifying Fc fragments of IgG's by affinity chromatography is
disclosed. The method comprising the steps of: (a) contacting a
sample comprising IgG Fc and at least one of IgG Fab or
F(ab').sub.2 fragments with a GB1 polypeptide of the present
invention immobilized to a solid phase support to immobilize the
IgG Fab or F(ab').sub.2 fragments to the solid phase support; and
(b) collecting the IgG Fc fragments remaining in the sample.
[0144] Optionally, the method can further comprise the step of
washing the solid phase support with a buffer of pH 5 to 8 to give
an eluate comprising Fc fragments. Examples of buffers having pH 5
to 8 include, but are not limited to acetate, phosphate, Tris,
borate, and bicarbonate.
[0145] The Fab or F(ab').sub.2 fragments bound to the immobilized
GB1 domain polypeptide may then be recovered by washing the solid
phase support with a suitable buffer, such as a buffer of about pH
5 to about pH 8 or a buffer of about pH 3.5 to about pH 2.4.
Examples of buffers having pH 5 to 8 include, but are not limited
to, acetate, phosphate, Tris, borate, and bicarbonate. Examples of
buffers having pH 3.5 to 2.4 include, but are not limited to,
acetate, citrate, and glycine.
[0146] Thus, in accordance with the present invention, a method for
purifying Fab and F(ab').sub.2 fragments of IgG's by affinity
chromatography is also disclosed. The method comprising the steps
of: (a) contacting a sample comprising IgG Fc and Fab fragments
with a GB1 polypeptide of the present invention immobilized to a
solid phase support to immobilize the IgG Fab fragments to the
solid phase support; (b) collecting the IgG Fc fragment remaining
in the sample; and (c) eluting the IgG Fab fragments from the solid
phase support to give purified IgG Fab fragments in the eluate.
Similar steps are performed with respect to F(ab').sub.2
fragments.
[0147] Elution of Fab or F(ab').sub.2 fragments may be accomplished
using art recognized techniques, such as pH gradients. For example,
it is recognized that protein A binds the Fc portion of IgG
antibodies across many mammalian species and may be used to purify
IgGs from a complex solution, cells coded with IgG, or Fc fragments
of IgG away from Fab fragments. The protocol is to flow the IgG
over a protein A-sepharose resin in a 20 mM sodium phosphate buffer
(pH 7.0) and to continue washing this buffer to remove non-specific
binding. The elution is performed with 20 mM sodium citrate (pH
4.0). Other acceptable elution techniques are described by
Jungpauer, A. et al. (1989) Journal of Chromatography 46:257.
[0148] J. Preferred Substances for use in Purification and
Detection Methods
[0149] Substances suitable for covalent or non-covalent coupling to
GB1 polypeptides include, but are not limited to, enzymes such as
horseradish peroxidase, alkaline phosphatase, acid phosphatase, and
luciferase; radioisotopes such as .sup.125I, .sup.35S, .sup.14C,
and .sup.3H; insoluble beads such as latex, agarose,
polyacrylamide, and magnetic particles; solid matrices such as
silica, and polystyrene; and colloidal metals such as gold.
Conjugation protocols are conventionally standardized for each type
of conjugate.
[0150] A variety of suitable vessels for incubation mixtures
containing antibodies and antibody-binding proteins and
polypeptides are available commercially. These include, but are not
limited to multiple sample vessels such as microtiter plates and
other tissue culture plasticware; vessels for western blot analysis
such as staining dishes, slot dishes, and plastic bags; materials
for immunofluorescence such as microscope slides, cover slips, and
sterile plasticware; chromatography materials such as disposable
chromatography columns, syringes, high performance and fast
performance chromatography columns; vessels for incubating reagents
in suspension such as test tubes, micro centrifuge tubes, beakers,
and flasks; and materials for electron microscopic sample
incubation such as Parlodion or Formvar-coated electron microscope
grids, and Parafllm.
[0151] Components preferred for the binding reaction between GB1
polypeptides and IgG's, fragments of an IgG, or combinations
thereof would include the components described above; a
physiological buffer compatible with the binding reaction such as
PBS, TBS or others listed above, or any other buffer which might
show applicability through further routine experimentation; and a
system for environmental temperature control capable of maintaining
the samples at temperatures ranging from 0.degree.-37.degree.
C.
[0152] Components preferred for the detection and/or visualization
of complexes between the GB1 polypeptides of the present invention
and an IgG, a fragment of an IgG, or combinations thereof, may
include but are not limited to the following: reagents for
chromogenic detection of enzyme conjugates such as enzyme
substrates, chromogenic dyes, and appropriate reaction buffers, a
mechanized calorimeter compatible with the reaction vessel is
advantageous for assays on numerous samples; materials for
detection of radiolabeled complexes such as gamma or scintillation
counters, X-ray film, and autoradiography cassettes; components for
fluorescent and illuminescent detection such as fluorescent
microscopes and ultraviolet light sources, fluorescence activated
cell sorter or flow cytometer, and film; and materials for IEM such
as buffers, metallic stains, and an electron microscope with
photographic capabilities.
[0153] Components preferred for the retention of IgG's, fragments
of an IgG, or combinations thereof, onto an insoluble matrix may
include, but are not limited to, insoluble conjugates of the GB1
polypeptides of the present invention as listed above.
[0154] Components for the recovery of IgG's, fragments of an IgG,
or combinations thereof, eluted from a complex between a GB1
polypeptide of the present invention and IgG's, fragments of an
IgG, or combinations thereof, may include but are not limited to:
materials for chromatographic purification systems such as
appropriate elution buffers, an ultraviolet absorbance detector, a
fraction collector, and containers or tubes for the collection of
eluted material; components for the recovery of IgG's, fragments of
an IgG, or combinations thereof, from suspended incubation mixtures
would include a dissociation buffer, centrifuge, and container for
collecting centrifugal supernatants.
[0155] Components preferred for the transfer, dehydration, or
concentration of purified IgG's, fragments of an IgG, or
combinations thereof, may require but are not limited to the
following: a dialysis system including dialysis membrane, a
volatile buffer or solvent desired for maintenance of the
antibodies such as those listed above, and a vessel in which to
dialyse the eluate; a vacuum concentrator or vacuum evaporator to
remove undesired quantities of solvent, containers to be
appropriate for storing resulting volumes of antibody preparations,
and antimicrobial preservative for maintenance of antibody
solutions (see above).
[0156] By the term "solid phase support" it is intended any support
capable of immobilizing a GB1 domain polypeptide of the invention,
either covalently, or by adsorption. Solid phase supports which may
be used for immobilizing the GB1 domain polypeptide of the
invention include, but are not limited to, polymers having hydroxyl
groups, either free or in esterified form, such as agarose,
cellulose, including cellulose esters such as cellulose nitrate,
diazocellulose, cellulose acetate, cellulose propionate, and the
like, and acrylamide polymers and copolymers, such as
polyacrylamide and acrylamide, microtitre plates, glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, agar,
starch, or the chemically active membrane having a large surface
area comprising a hydrophobic, microporous, skinless, polyamide
membrane which is chemically bound to a residue of an activating
agent which is capable of immobilizing the GB1 domain polypeptide
of the invention. See Degen et al. (1987) U.S. Pat. No. 4,693,985,
herein incorporated by reference.
[0157] The GB1 domain polypeptide may be immobilized to the solid
phase support according to methods known to those of ordinary skill
in the art for protein immobilization. For example, the GB1 domain
polypeptide may be coated or bonded, either covalently or by
adsorption, to the solid phase. Methods for immobilizing proteins
to solid phase supports are taught, for example, in U.S. Pat. Nos.
3,652,761, 3,879,262, 3,986,217, and 4,693,985, the contents of
each of which herein incorporated by reference. Preferably, the GB1
domain polypeptides are immobilized to tresyl activated or cyanogen
bromide activated agarose.
LABORATORY EXAMPLES
[0158] The following Laboratory Examples have been included to
illustrate preferred modes of the invention. Certain aspects of the
following Laboratory Examples are described in terms of techniques
and procedures found or contemplated by the present inventors to
work well in the practice of the invention. These Laboratory
Examples are exemplified through the use of standard laboratory
practices of the inventors. In light of the present disclosure and
the general level of skill in the art, those of skill will
appreciate that the following Laboratory Examples are intended to
be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
MATERIALS AND METHODS USED IN THE EXAMPLES
[0159] Mutagenesis. The construction of the B1 domain construct
(Q32C) was performed as reported by Sloan. D. J. and Hellinga. H.
W. (1998) Prot Eng 11:819-823. All mutations in this study were
constructed in that background by PCR mutagenesis as described by
Ho. S. N. et al. (1989) Gene 77:55-59). A C-terminal
oligo-histidine (His.sub.5) affinity tag was used to allow the
purification of the mutant proteins by immobilized metal affinity
chromatography as described by Hochuli. E. et al. (1988)
Bio/Technology 6:1321-1325. The recombinant genes were flanked by
EcoRI sites that allowed facile cloning into either the pKK223-3
vector for protein expression or the M13 construct for phage
display using gill fusions.
[0160] Protein Expression and Purification. The expression and
purification of mutant B1 domains was performed as described by
Sloan, D. J. and Hellinga. H. W. (1998) Prot. Eng. 11:819-823.
Briefly, the B1 domain proteins were expressed in the prokaryotic
expression vector pKK223-3 (GenBank Accession Number M77749).
Pkk223-3 is sold by Amersham-Pharmacia, Uppsala, Sweden. Protein
expression is controlled by the Tac promoter. pKK223-3 is grown in
the bacterial (E. coli) strain XL1-Blue (purchased from Stratagene,
La Jolla, Calif.) which expresses the lac repressor allowing for
inducible productiuon of the B1 domain proteins. This vector is
also described in more detail by Brosius, J. et al. (1981) Plasmid
6:112-118 and Brosius. J. et al. (1981) J. Mol. Biol. 148:107-127.
Atypical expression experiment produced 15 milligrams of pure
mutant protein from 1 liter of culture.
[0161] Fluorophore coupling and iodoacetamide blocking. All of the
above mutations were constructed into the Q32C background to allow
site-specific covalent coupling with acrylodan for fluorescent
binding assays as described by Sloan, D. J. and Hellinga, H. W.
(1998) Prot. Eng. 11:819-823. In competitive binding experiments
between fluorescently labeled wild-type B1 and unlabeled mutants,
the free thiol in the mutant protein was blocked by reaction with
iodoacetamide.
[0162] Binding of B1 mutants to human IgG Fc fragment. The binding
constants for each of the alanine mutants were measured as an
increase in fluorescence by direct titration of Fc fragment of
human IgG (ICN Biomedicals, Costa Mesa, Calif.) into a solution
containing 250 nM acrylodan-conjugated B1 domain mutant in 20 mM
KPO.sub.4 (pH 6.0) at 25.degree. C. (excitation 392 nm; emission
500 nm; slit widths of 4 and 16 nm respectively). After each
addition of protein, the solution was allowed to equilibrate for 30
seconds before the final fluorescence value was recorded.
[0163] Each titration comprised 20 points and was performed in
triplicate. The data were fit to a binding isotherm that described
a single binding site and takes into account all of the species
present (Segel. I. H. (1975) Enzyme Kinetics, John Wiley &
Sons, New York, N.Y.): 1 F = F 0 + ( F max - F 0 ) ( [ B 1 ] t + [
h F c ] t + K d ) - ( [ B 1 ] t + [ h F c ] t + K d ) 2 - 4 [ B 1 ]
t [ h F c ] t 2 [ B 1 ] t
[0164] where F is the measured fluorescence with F.sub.0 and
F.sub.max representing the initial and final values, respectively.
[B1].sub.t and [hFc].sub.t are the concentrations of B1 and hFc
respectively, and the K.sub.d is the disassociation constant. This
equation is referred to herein as "Equation 1".
[0165] The binding constants of weak binders (greater than 50-fold
increases in K.sub.d) were measured in a competition assay with a
pre-formed complex (250 nM) of wild type Q32C construct conjugated
to acrylodan and hFc. Binding constants were determined by
measuring the decrease in fluorescence of the complex by the
addition of iodoacetamide blocked mutant B1 domains. From these
competition binding curves the K.sub.d for each of the mutant B 1
domains was calculated from the binding isotherm (Fierke, C. A. et
al. (1991) Biochemistry 30:11054-11063): 2 F = F 0 1 + ( K D WT [ B
1 w t ] ) .times. ( 1 + [ B 1 mut ] K Dmut ) + F f
[0166] where F is the measured fluorescence with F.sub.0 and
F.sub.t representing the initial and final values respectively.
[B1.sub.wt] and [B1.sub.mut] are the concentrations of B1wild type
and mutant respectively, and the K.sub.d is the disassociation
constant of the mutant or wild type. This Equation is referred to
herein as "Equation 2".
[0167] Phage Display. A gly-gly-gly-ser-gly-gly-gly-ser linker was
inserted into the NotI site of pCANTAB5 (Pharmacia, Bridgewater,
N.J.) to reduce the interactions between the displayed protein and
PIII in accordance with techniques disclosed by Smith. G. P. and
Scoft, J. K. (1993) Methods Enzymol. 217:228-257. The resulting
gill gene fusion was sub-cloned from pCANTAB5 into the multiple
cloning site of M13 mp18 (Messing, J. (1991) Gene 100:3-12). A
unique NheI site was introduced into the gill fusion by
site-directed mutagenesis as described by Kunkel, T. A. (1985)
Proc. Natl. Acad. Sci. USA 82:488-492) into which the B1 gene was
cloned. Alanine mutations were constructed directly into this
vector.
[0168] Biopanning as described by Smith, G. P. and Scoft, J. K.
(1993) Methods Enzymol. 217:228-257 was used to determine the
Fab-binding properties of the fusion constructs, using 100 ng
biotinylated IgG, 5.times.1 09 pfu of each B1 bacteriophage, high
protein binding ELISA wells (Greiner, Kremsmunster, Austria), and
2% non-fat dry milk blocking solution. Binding between mutant B1
and IgG was detected by anti-M13 antibody-HRP conjugate (Pharmacia,
Bridgewater, N.J.; manufacturers protocol). Control experiments in
which the Fab fragment was left out were done in parallel to rule
out non-specific binding.
[0169] Table 3 presents the nucleic acid and peptide sequences of
the naturally occurring or native GB1 polypeptide including a
methionine residue at amino acid residue -1 which was incorporated
through recombinant DNA techniques to facilitate expression of the
polypeptide in the expression vector system disclosed herein. Table
4 summarizes the Examples, which comprises the mutant GB1
polypeptides of the present invention.
4TABLE 3 Nucleic Acid and Peptide Sequences of Native B1 Domain of
Protein G (SEQ ID No:1 and SEQ ID NO:2) ATG ACT ACT TAC AAA TTA ATC
CTT AAT GGT AAA ACA TTG AAA GGC GAA ACA MET THR THR TYR LYS LEU ILE
LEU ASN GLY LYS THR LEU LYS GLY GLU THR ACT ACT GAA GCT GTT GAT GCT
GCT ACT GCA GAA AAA GTG TTC AAA CAA TAC THR THR GLU ALA VAL ASP ALA
ALA THR ALA GLU LYS VAL PHE LYS GLN TYR GCT AAC GAG AAC GGT GTT GAC
GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ALA ASN ASP ASN GLY VAL ASP
GLY GLU TRP THR TYR ASP ASP ALA THR LYS ACC TTT ACA GTT ACT GAA CAT
CAC CAT CAT CAC TAA GCT TGA THR PHE THR VAL THR GLU HIS HIS HIS HIS
HIS OCH ALA OPA Met is residue-1 and the start site of translation
in the expression vector system disclosed herein. Underlined
residues are the polyhistidine tag used for purification in
immobilized metal affinity chromatography. Italicized are two stop
codons used to terminate translation. # Shown in bold is the
crucial residue glutamate 27 which when mutated to alanine (GCG) or
to any other amino acid abolishes binding to the Fc fragment of IgG
while retaining binding to Fab fragments. Shown in italics and
underlining are other residues described in Table 3 which when
mutated to alanine (GCG) substantially abolish binding to the Fc
fragment of IgG while retaining binding to Fab fragments. In
accordance with the present invention other amino acids can be
substituted for these residues, including for example, valine,
leucine and isoleucine. Codons for such substitutions can be
selected from Table 2.
[0170]
5TABLE 4 Ex- am- K.sub.d.sup.a DDG.sup.b SEQ ID ple Mutant (mM)
(kcal/mole) H-bonds.sup.c f.sub.SASA.sup.d NO 1 Q32C.sup.e 0.24 0 1
0.53 3 2 T25A 0.36 0.24 0 0.6 4 3 E27A >1000.sup.f >4.9.sup.f
3 1 6 4 K28A 2.0 1.3 1 0.95 8 5 K31A 85 3.5 0 0.94 10 6 N35A 13 2.4
2 0.79 12 7 D40A 0.38 0.3 0 0.40 13 8 E42A 0.46 0.4 1 0.30 14 9
W43A 140 3.8 1 0.89 16 10 T44/ 6.5 2.0 0 0.43 18 Y45A .sup.aK.sub.d
calculated from Equation 1. .sup.bDDG = RTln (K.sub.d wild
type/K.sub.d mutant); T = 298K. .sup.cNumber of hydrogen bonds
formed by the native, non-mutated residue and the Fc as determined
by inspection of the crystal structure of FIG. 1. .sup.df.sub.SASA
is fractional change in solvent accessible surface area: f.sub.SASA
= SASA(complex)/SASA(free) .sup.eThe wild-type construct relative
to which all measurements are made. .sup.fBinding is too weak to be
quantified.
RESULTS OF EXAMPLES
[0171] Mutagenesis of the Fc-binding site on the B1 domain. The
X-ray structure of the complex between the B1 and a human IgG Fc
fragment (hFc) depicted schematically in FIG. 1 and as described by
Sauer-Eriksson. A. E. et al. (1995) Structure 3:265-278 was used to
identify the residues located in the interface between these two
proteins. The static solvent-accessible surface (Richards, F. M.
(1977) Annual Review of Biophysics and Bioengineering 6:151-176)
was calculated for the B1 domain in the presence and absence of the
hFc fragment. Eleven residues showed a significant decrease in
solvent accessibility in the structure of the complex (Table 4).
Single alanine mutants of each of these residues were constructed.
These are located in the alpha helix, the third P strand, and the
loop between this helix and strand (FIG. 1).
[0172] Binding studies. The interaction of the mutant B1 domain
with the hFc was measured using a fluorescence method described by
Sloan. D. J. and Hellinga. H. W. (1998) Prot. Eng. 11:819-823,
which relies on the attachment of an environmentally sensitive
fluorophore placed at the rim of the binding site in a location
such that its fluorescence changes upon formation of the complex by
virtue of alterations in conformational degrees of freedom and
solvent accessibility, without adversely affecting the binding
constant. Binding constants for hFc were determined in a direct
titration experiment in which purified hFc was added to a solution
of a Q32C B1 mutant to which the fluorophore acrylodan was
covalently coupled at position 32C (FIG. 2A). The binding constants
for all the B1 mutants were determined in the Q32C background
(Table 4).
[0173] For those mutants whose K.sub.d showed a greater than
fifty-fold increase, the binding constants were determined in a
competition experiment in which a complex was preformed between
fluorescently labeled wild-type B1 (Q32C) and hFc, which was then
titrated with an unlabeled, mutant B1 domain whose free cysteine at
position 32 had been blocked with iodoacetamide (FIG. 2B). This
approach was used to avoid having to use large quantities of pure
hFc.
[0174] In order to verify that large decreases in the binding
constants were not due to loss of structure, we took advantage of
the fact that B1 has a separate binding site for the Fab fragment,
which does not overlap with the Fc binding site. Fab binding was
determined semi-quantitatively by phage display in M13 as described
by Smith, G. P. and Scott. J. K. (1993) Methods Enzymol. 217:
228-257 using a genetic fusion between gene III and B1, expressed
as a second copy in the phage genome, as described by Armstrong. N.
et al. (1996) in Phage display of peptides and proteins (Kay, B.
K., Winter, J., & McCafferty, J., Eds.) pp. 35-53, Academic
Press, Inc., San Diego, Calif., in order to obtain uni-valent
rather than poly-valent display of the B1 domain. See also
McConnell. S. J. (1994) Gene 151:115-118. In all cases, the mutants
constructed in the Fc-binding site retained the abilityto bind Fab,
indicating that there was no significant loss in overall
structure.
[0175] The results show that five out of the ten residues in the
binding site had a significant effect on hFc binding (K.sub.d
increased at least ten-fold), whereas the other five showed little
or no effect (less than two-fold change in K.sub.d). A single
mutant, E27A, virtually abolished binding (>4000-fold increase
in Kd). The five residues that most affected binding form a
contiguous patch on the surface of B1, surrounded by a ring of the
other residues (FIG. 1).
DISCUSSION OF EXAMPLES
[0176] The experiments described herein report the energetic
contributions of the residues forming the binding site between the
B1 domain of protein G and human IgG Fc fragment. The X-ray
structure of a complex between the B1 domain and hFc show that
about thirteen residues on the surface of B1 are directly involved
in the binding site as defined by a change in their
solvent-accessible area upon complex formation. Three of these
residues are alanines and glycine scanning mutagenesis was not
performed to determine the energetic contribution of these
residues. Alanine mutations of only about half of the ten mutated
residues significantly affect the binding free energy (at least
ten-fold increase in K.sub.d. Within these five residues the effect
of binding varies sharply, ranging from 10-fold to more than
4000-fold increases in K.sub.d, indicating that the determinants
contributing to the free energy of binding are highly localized.
The localization of binding energy to such "hot-spots" has also
been observed in other heterodimeric interfaces (Bogan A. A. and
Thorn K. S. (1998) J. Mol. Biol. 280:1-9); (Wells, J. A. (1991)
Methods Enzymol. 202:390-411).
[0177] The binding hot-spots on the surface of the B1 domain are
associated with clear structural motifs. The binding site is formed
by two "knobs-into-holes" interactions. E27 of the B1 domain fits
into a hole formed by 1253 and S254 on the surface of the Fc
fragment, where the carboxylate forms hydrogen bonds with the
backbone amides of these residues as well as the O.sub.y of S254.
Mutation of this charged knob on the surface of the B1 domain
virtually completely abolishes binding. The carboxylate of E27 is
held in position by a hydrogen bond from the amino group of K31,
the neighboring residue, on the surface of B1. Removal of this
interaction in the K31A mutant significantly decreases affinity
(350-fold increase in K.sub.d). The second knob-into-hole
interaction is formed by the protrusion of N434 from the surface of
the Fc into a hole in B1 bordered by N35, D36, D40, E42 and W43.
The indole nitrogen of W43 forms a hydrogen bond with N434. Of all
the bordering residues forming the hole on B1, the W43A mutant has
the most profound effect on the interaction (580-fold increase in
K.sub.d), presumably as a direct consequence of this interaction.
Of the other residues, only N35A significantly affects binding
(50-fold increase in K.sub.d). Interestingly, it makes a hydrogen
bond with H433 on the hFc.
[0178] These results quantify the contribution of the individual
residues identified in the high-resolution structure of the B1-hFc
complex (Sauer-Eriksson, A. E. et al. (1995) Structure 3:265-278).
It was noted in the description of this structure that all of these
residues are involved in contacts between the two proteins. From
this study it is clear, however, that only a small subset make
significant contribution to the free energy of binding,
illustrating the importance of combining structural information
with thermodynamic data. It should be emphasized that in an
alanine-scanning mutagenesis experiment side-chains are deleted.
Lack of a large effect upon loss of an interaction does not imply
that the choice of a particular amino acid is unimportant, since
replacement with residues which result in incorrect steric packing
or charge complementarity may adversely affect proper formation of
the interface. Furthermore, an alanine scanning experiment does not
address the issue of main-chain interactions. Two B1 domain
residues potentially form main chain interactions with hFc
residues, those being the nitrogen of G41 and the carbonyl oxygen
of V39 (Sauer-Eriksson, A. E. et al. (1995) Structure
3:265-278).
[0179] The four critical binding residues within the B1 domain are
glutamate, tryptophan, lysine, and asparagine. Tryptophan is
frequently found within interfaces, whereas lysine and asparagine
are not normally enriched, and glutamate is underrepresented in
heterodimer interfaces. See Bogan A. A. and Thorn K. S. (1998) J.
Mol. Biol. 280:1-9. It has been suggested that hot spots located in
planar interfaces need to be surrounded by a ring of residues that
exclude bulk solvent (the O-ring hypothesis), aiding in the
formation of polar hydrogen bonds (Bogan A. A. and Thorn K. S.
(1998) J. Mol. Biol. 280:1-9). The polar knobs-into-holes binding
motif observed in the B1 domain provides another mechanism to form
buried, polar hydrogen bonds.
[0180] The existence of a well-defined hot-spot suggests that it
may be possible to develop a low-molecular weight reagent that
could disrupt this interface. Such a reagent could have
applications in immunochemistry as well in treatment of
streptococcal infections by potentially unmasking bacteria cloaked
in a coat of antibodies.
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[0181] The references listed below as well as all references cited
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extent that they supplement, explain, provide a background for or
teach methodology, techniques and/or compositions employed
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[0188] Bodanszky et al. (1976) "Peptide Synthesis", John Wiley
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280:1-9.
[0190] Boyle, M. D. P. (1990) Bacterial Immunoglobulin-binding
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[0254] It will be understood that various details of the invention
may be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
Sequence CWU 1
1
24 1 195 DNA Streptococcus sp. CDS (-1)..(195) 1 atg act act tac
aaa tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca
act act gaa gct gtt gat gct gct act gca gaa aaa gtc ttc aaa 96 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25
30 caa tac gct aac gac aac ggt gtt gac ggt gaa tgg act tac gac gat
144 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac
taa gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His
Ala 50 55 60 tga 195 2 62 PRT Streptococcus sp. 2 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25
30 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50
55 60 3 62 PRT Streptococcus sp. 3 Met Thr Thr Tyr Lys Leu Ile Leu
Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr Glu Ala
Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30 Cys Tyr Ala
Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40 45 Ala
Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55 60 4 62
PRT Streptococcus sp. 4 Met Thr Thr Tyr Lys Leu Ile Leu Asn Gly Lys
Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr Glu Ala Val Asp Ala
Ala Ala Ala Glu Lys Val Phe Lys 20 25 30 Gln Tyr Ala Asn Asp Asn
Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40 45 Ala Thr Lys Thr
Phe Thr Val Thr Glu His His His His His 50 55 60 5 195 DNA
Streptococcus sp. CDS (-1)..(195) 5 atg act act tac aaa tta atc ctt
aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr Lys Leu Ile Leu
Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca act act gaa gct
gtt gat gct gct act gca gcg aaa gtc ttc aaa 96 Thr Thr Thr Glu Ala
Val Asp Ala Ala Thr Ala Ala Lys Val Phe Lys 20 25 30 caa tac gct
aac gac aac ggt gtt gac ggt gaa tgg act tac gac gat 144 Gln Tyr Ala
Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40 45 gcg
act aag acc ttt aca gtt act gaa cat cac cat cat cac taa gct 192 Ala
Thr Lys Thr Phe Thr Val Thr Glu His His His His His Ala 50 55 60
tga 195 6 62 PRT Streptococcus sp. 6 Met Thr Thr Tyr Lys Leu Ile
Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr Glu
Ala Val Asp Ala Ala Thr Ala Ala Lys Val Phe Lys 20 25 30 Gln Tyr
Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40 45
Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55 60 7
195 DNA Streptococcus sp. CDS (-1)..(195) 7 atg act act tac aaa tta
atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr Lys Leu
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca act act
gaa gct gtt gat gct gct act gca gaa gcg gtc ttc aaa 96 Thr Thr Thr
Glu Ala Val Asp Ala Ala Thr Ala Glu Ala Val Phe Lys 20 25 30 caa
tac gct aac gac aac ggt gtt gac ggt gaa tgg act tac gac gat 144 Gln
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40
45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac taa gct
192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His Ala 50
55 60 tga 195 8 62 PRT Streptococcus sp. 8 Met Thr Thr Tyr Lys Leu
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr
Glu Ala Val Asp Ala Ala Thr Ala Glu Ala Val Phe Lys 20 25 30 Gln
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35 40
45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55 60
9 195 DNA Streptococcus sp. CDS (-1)..(195) 9 atg act act tac aaa
tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr Lys
Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca act
act gaa gct gtt gat gct gct act gca gaa aaa gtc ttc gcg 96 Thr Thr
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Ala 20 25 30
caa tac gct aac gac aac ggt gtt gac ggt gaa tgg act tac gac gat 144
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35
40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac taa
gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His Ala
50 55 60 tga 195 10 62 PRT Streptococcus sp. 10 Met Thr Thr Tyr Lys
Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Ala 20 25 30
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp 35
40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55
60 11 195 DNA Streptococcus sp. CDS (-1)..(195) 11 atg act act tac
aaa tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca
act act gaa gct gtt gat gct gct act gca gaa aaa gtc ttc aaa 96 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25
30 caa tac gct gcg gac aac ggt gtt gac ggt gaa tgg act tac gac gat
144 Gln Tyr Ala Ala Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac
taa gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His
Ala 50 55 60 tga 195 12 62 PRT Streptococcus sp. 12 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25
30 Gln Tyr Ala Ala Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50
55 60 13 62 PRT Streptococcus sp. 13 Met Thr Thr Tyr Lys Leu Ile
Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr Glu
Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30 Gln Tyr
Ala Asn Asp Asn Gly Val Ala Gly Glu Trp Thr Tyr Asp Asp 35 40 45
Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55 60 14
62 PRT Streptococcus sp. 14 Met Thr Thr Tyr Lys Leu Ile Leu Asn Gly
Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr Glu Ala Val Asp
Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30 Gln Tyr Ala Asn Asp
Asn Gly Val Asp Gly Ala Trp Thr Tyr Asp Asp 35 40 45 Ala Thr Lys
Thr Phe Thr Val Thr Glu His His His His His 50 55 60 15 195 DNA
Streptococcus sp. CDS (-1)..(195) 15 atg act act tac aaa tta atc
ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr Lys Leu Ile
Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca act act gaa
gct gtt gat gct gct act gca gaa aaa gtc ttc aaa 96 Thr Thr Thr Glu
Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30 caa tac
gct aac gac aac ggt gtt gac ggt gaa gcg act tac gac gat 144 Gln Tyr
Ala Asn Asp Asn Gly Val Asp Gly Glu Ala Thr Tyr Asp Asp 35 40 45
gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac taa gct 192
Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His Ala 50 55
60 tga 195 16 62 PRT Streptococcus sp. 16 Met Thr Thr Tyr Lys Leu
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr Thr
Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30 Gln
Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Ala Thr Tyr Asp Asp 35 40
45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55 60
17 195 DNA Streptococcus sp. CDS (-1)..(195) 17 atg act act tac aaa
tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr Lys
Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca act
act gaa gct gtt gat gct gct act gca gaa aaa gtc ttc aaa 96 Thr Thr
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30
caa tac gct aac gac aac ggt gtt gac ggt gaa tgg gcg gcg gac gat 144
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Ala Ala Asp Asp 35
40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac taa
gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His Ala
50 55 60 tga 195 18 62 PRT Streptococcus sp. 18 Met Thr Thr Tyr Lys
Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr Thr
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys 20 25 30
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Ala Ala Asp Asp 35
40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50 55
60 19 195 DNA Streptococcus sp. CDS (-1)..(195) 19 atg act act tac
aaa tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 aca
act act gaa gct gtt gat gct gct act gca gtt aaa gtc ttc aaa 96 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Val Lys Val Phe Lys 20 25
30 caa tac gct aac gac aac ggt gtt gac ggt gaa tgg act tac gac gat
144 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat cac
taa gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His
Ala 50 55 60 tga 195 20 62 PRT Streptococcus sp. 20 Met Thr Thr Tyr
Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15 Thr
Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Val Lys Val Phe Lys 20 25
30 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His His 50
55 60 21 195 DNA Streptococcus sp. CDS (-1)..(195) 21 atg act act
tac aaa tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met Thr Thr
Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15
aca act act gaa gct gtt gat gct gct act gca tta aaa gtc ttc aaa 96
Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Leu Lys Val Phe Lys 20
25 30 caa tac gct aac gac aac ggt gtt gac ggt gaa tgg act tac gac
gat 144 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
Asp 35 40 45 gcg act aag acc ttt aca gtt act gaa cat cac cat cat
cac taa gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His
His Ala 50 55 60 tga 195 22 62 PRT Streptococcus sp. 22 Met Thr Thr
Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5 10 15
Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Leu Lys Val Phe Lys 20
25 30 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
Asp 35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His His His
His 50 55 60 23 195 DNA Streptococcus sp. CDS (-1)..(195) 23 atg
act act tac aaa tta atc ctt aat ggt aaa aca ttg aaa ggc gaa 48 Met
Thr Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5
10 15 aca act act gaa gct gtt gat gct gct act gca att aaa gtc ttc
aaa 96 Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Ile Lys Val Phe
Lys 20 25 30 caa tac gct aac gac aac ggt gtt gac ggt gaa tgg act
tac gac gat 144 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr
Tyr Asp Asp 35 40 45 gcg act aag acc ttt aca gtt act gaa cat cac
cat cat cac taa gct 192 Ala Thr Lys Thr Phe Thr Val Thr Glu His His
His His His Ala 50 55 60 tga 195 24 62 PRT Streptococcus sp. 24 Met
Thr Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu -1 1 5
10 15 Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Ile Lys Val Phe
Lys 20 25 30 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr
Tyr Asp Asp 35 40 45 Ala Thr Lys Thr Phe Thr Val Thr Glu His His
His His His 50 55 60
* * * * *
References