U.S. patent application number 13/139332 was filed with the patent office on 2011-11-10 for yeast display systems.
This patent application is currently assigned to NOVARTIS AG. Invention is credited to Andreas Loew.
Application Number | 20110275535 13/139332 |
Document ID | / |
Family ID | 42115977 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110275535 |
Kind Code |
A1 |
Loew; Andreas |
November 10, 2011 |
Yeast Display Systems
Abstract
The present invention relates to the field of protein display
libraries and library screening. In preferred embodiments, the
present invention provides a three component system for display
comprising a cell surface molecule, an adapter molecule and a
display molecule.
Inventors: |
Loew; Andreas; (Cambridge,
MA) |
Assignee: |
NOVARTIS AG
Basel
CH
|
Family ID: |
42115977 |
Appl. No.: |
13/139332 |
Filed: |
December 14, 2009 |
PCT Filed: |
December 14, 2009 |
PCT NO: |
PCT/EP2009/067066 |
371 Date: |
June 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61122910 |
Dec 16, 2008 |
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Current U.S.
Class: |
506/9 ; 506/14;
506/26 |
Current CPC
Class: |
C07K 2319/035 20130101;
C07K 2319/70 20130101; C40B 40/02 20130101; C12N 15/815 20130101;
C07K 14/78 20130101; C07K 14/395 20130101; C40B 50/06 20130101;
C12N 15/81 20130101; C12N 15/1037 20130101; C07K 2319/912 20130101;
C40B 30/04 20130101; C40B 40/08 20130101 |
Class at
Publication: |
506/9 ; 506/14;
506/26 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 50/06 20060101 C40B050/06; C40B 40/02 20060101
C40B040/02 |
Claims
1. A library of host cells, wherein each host cell comprising: (a)
a cell surface molecule attached to the surface of the cell, (b) an
adapter molecule comprising a first binding site and a second
binding site, and (c) a display molecule comprising a modified
polypeptide; wherein the first binding site binds specifically to
the cell surface molecule and cannot bind to the display molecule
and the second binding site binds specifically to the display
molecule and cannot bind to the cell surface molecule, and wherein
the adapter molecule is not a component of the modified
polypeptide.
2. The host cells of claim 1, further comprising a plurality of
display molecules.
3. The host cells of claim 1, wherein the host cell surface
molecule is covalently linked to the first binding site.
4. The host cells of claim 1, wherein the host cell surface
molecule is covalently linked to the first binding site through a
disulfide bond.
5. The host cells of claim 1, wherein the host cell surface
molecule comprises a first agglutinin that is Aga1p, and wherein
the first binding site comprises a second agglutinin that is
Aga2p.
6. (canceled)
7. The host cells of claim 1, wherein the host cell surface
molecule is attached to the cell membrane via a GPI anchor.
8. (canceled)
9. (canceled)
10. The host cells of claim 1, wherein the second binding site is
covalently linked to the display molecule.
11. The host cells of claim 1, wherein the second binding site is
covalently linked to the display molecule through disulfide
bonds.
12. The host cells of claim 1, wherein the second binding site
comprises a PDZ domain of InaD.
13. (canceled)
14. The host cells of claim 12, wherein the display molecule
comprises a C-terminal NorpA ligand.
15. The host cells of claim 1, wherein the display molecule
comprises a PDZ domain of InaD.
16. (canceled)
17. The host cells of claim 15, wherein the second binding site
comprises a C-terminal NorpA ligand.
18. The host cells of claim 1, wherein the modified polypeptide is
selected from the group consisting of: a scaffold protein, a signal
transduction protein, an antibody, an immunoglobulin, an
immunoadhesin, a receptor, a ligand, an oncoprotein, a
transcription factor, an enzyme, and a fibronectin polypeptide.
19. The host cells of claim 18, wherein the display molecule is a
fibronectin polypeptide.
20. The host cells of claim 19, wherein the fibronectin polypeptide
comprises an F10 polypeptide.
21. The host cells of claim 1, wherein the display molecule
comprises a secretion signal peptide.
22. The host cells of claim 21, wherein the secretion signal
peptide comprises an MFalpha/HSA hybrid leader peptide, or an
MFalpha leader peptide.
23. (canceled)
24. The host cells of claim 1, wherein expression of the display
molecule is under the control of a first inducible promoter
selected from the group consisting of an AOX1 promoter, a CUP 1
promoter, and a Gal 1 promoter.
25. (canceled)
26. (canceled)
27. The host cells of claim 1, wherein the expression of the
adapter molecule is under the control of a second inducible
promoter selected from the group consisting of an AOX1 promoter, a
CUP 1 promoter, and a Gal 1 promoter.
28. (canceled)
29. (canceled)
30. The host cells of claim 1, wherein the host cell is a yeast
cell.
31. (canceled)
32. (canceled)
33. A method for displaying a modified polypeptide comprising: (a)
providing a host cell comprising a cell surface molecule attached
to the surface of the cell and a first nucleic acid encoding a
display polypeptide comprising a modified polypeptide, (b)
contacting the host cell with an adapter molecule comprising a
first binding site and a second binding site under conditions
wherein the first binding site binds to the cell surface molecule,
and (c) incubating the host cell under conditions wherein the host
cell exports the display polypeptide outside the host cell under
conditions wherein the second binding site binds to the display
polypeptide, wherein the first binding site binds specifically to
the cell surface molecule and cannot bind to the display molecule
and the second binding site binds specifically to the display
polypeptide and cannot bind to the cell surface molecule, and
wherein the adapter molecule is not a component of the modified
polypeptide.
34. The method of claim 33, wherein the host cell displays at least
10.sup.2, at least 10.sup.3, at least 10.sup.4, or at least
10.sup.5 modified polypeptides.
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (cancelled)
55. (canceled)
56. (canceled)
57. (canceled)
58. (cancelled)
59. (canceled)
60. (canceled)
61. (canceled)
62. (canceled)
63. (canceled)
64. A method for generating a host cell display library comprising:
introducing into a plurality of host cells a display library of
first nucleic acids each encoding a display polypeptide comprising
a modified polypeptide, wherein at least two of the introduced
first nucleic acids encode different modified polypeptides, wherein
each host cell comprises a second nucleic acid which encodes a cell
surface polypeptide and a third nucleic acid which encodes an
adapter molecule comprising a first binding site and a second
binding site, wherein the first binding site binds to the cell
surface molecule but not the display polypeptide and the second
binding site binds to the display polypeptide but not the cell
surface molecule, and wherein the adapter molecule is not a
component of the modified polypeptide.
Description
FIELD
[0001] The present invention relates to the field of protein
display libraries and library screening. More specifically, the
present invention relates to the production of proteins for display
on cell surfaces.
BACKGROUND
[0002] Protein binding domains can be predicted from sequence data,
however re-designing proteins with improved or altered binding
affinities often requires testing of a number of variants of the
re-designed protein. Currently the best method for obtaining
proteins with desired binding affinities is to generate and screen
a protein library including such variants that can include
rationally redesigned proteins, randomly altered proteins, or a
combination thereof. Libraries of many types of protein, such as
immunoglobulins and scaffold proteins and receptors or receptor
ligands have successfully been constructed and screened for binding
affinity.
[0003] There are many methods to screen libraries, but one of the
most common methods is the phage display method, which comprises
fusion of a protein library to the coat proteins of filamentous
phage e.g.,(Huse et al., '89; Clackson et al,, '91); Marks et al.,
'92). Fusions are made most commonly to a minor coat protein,
called the gene III protein (pIII), which is present in three to
the copies at the tip of the phage. The fused library is then
displayed on the surface of the viral particle. The pap display
library can then be screened against an immobilized target protein.
However, one major drawback of this method is that target proteins
that bind the library with very high affinity are not always
identified because the conditions required to elute the bound phase
usually denature the phage particle such that it becomes impossible
to identify the protein of interest. Another draw back of phage
display libraries is the requirement that the target protein be
immobilized on a solid surface, which can lead to difficulties in
determining the actual affinity of a target protein for the phage
display protein. Furthermore, some proteins of interest require
post-translational modifications, such as glycosylation,
methylation, or disulfide binding, that cannot be achieved when
expressed in a phage particle.
[0004] An alternative method for screening protein libraries is to
display the library on the surface of bacterial cells. This method
solves many of the drawbacks associated with phage display but has
its own problems. One problem with bacterial display is that the
bacterial capsule can cause steric hindrance to proteins displayed
on the bacterial surface. Also, bacteria do not contain the
machinery to properly fold eukaryotic proteins, so the protein of
interest may not always be expressed within the bacterium. Similar
to the problem in phage, bacteria cannot provide post-translational
modifications, like disulfide binding, to a eukaryotic protein.
[0005] Wittrup et al. (U.S. Pat. Nos. 6,699,558 and 5,696,251) have
developed a method for a yeast cell display library. This is a two
component system, wherein the first component involves expressing
one subunit of the yeast mating adhesion protein, agglutinin, which
is anchored to the yeast cell wall. The second component involves
expressing a protein library fused to a second subunit of the
agglutinin protein which forms high affinity disulfide bonds to the
first agglutinin subunit. The protein library fused to the
agglutinin is thus displayed on the surface of the cell. The
library can then be screened. This method allows for the proper
folding and post-translational modification of eukaryotic
proteins.
[0006] Rakestraw et al. (PCT/US20081003978) have developed a three
component system for displaying a protein library on the surface of
yeast cells. The first component involves expressing a protein
library fused to a biotin-binding peptide, the second component
involves modifying the yeast cell wall to express biotin, and the
third component involves binding avidin to the biotin expressed on
the cell surface. The fused protein library is then biotinylated
and secreted from the yeast cell and binds to the avidin on the
yeast cell surface, thus displaying the protein library on the
surface of the yeast cell. One potential drawback of this system is
that avidin non-specifically binds all biotin. Another potential
drawback is that avidin contains four binding sites, which may
cause steric hindrance thus preventing the biotinylated protein
library from binding to the cell surface bound avidin. Similarly,
the avidin molecule may bind the biotin) fated protein library
before binding the biotinylated yeast cell wall, thereby hindering
the binding of the avidin to the yeast cell wall. Additionally,
this method contains the added complication of having to
biotinylate the protein library within the yeast cell. This
necessary extra step requires further modification to the yeast
cell. It is well established that the more modifications are made
to a biological system, the less likely it is that the system will
behave as designed. In addition, since avidin/streptavidin is
multivalent, care must be taken to not cross-link the biotinylated
cells. Finally, biotin/streptavidin and biotin/avidin are used in a
number of commercially available labeling kits. Such kits would be
difficult to use in such a biotin/avidin display system.
[0007] The prior art lacks a simple, efficient system capable of
specifically binding a secreted protein library using an adapter
molecule that binds to the protein library and to the surface of a
eukaryotic cell through different binding moieties.
SUMMARY OF THE INVENTION
[0008] The present invention meets this need by providing methods
and compositions as disclosed through out the specification. One
aspect includes host cells with a cell surface molecule attached to
the surface of the cell, an adapter molecule comprising a first
binding site and a second binding site and a display molecule
comprising a modified polypeptide where the first binding site
binds specifically to the cell surface molecule and cannot bind to
the display molecule, the second binding site binds specifically to
the display molecule and cannot bind to the cell surface molecule,
and adapter molecule is not a component of the modified
polypeptide. In certain embodiments, the host cell has a plurality
of display molecules. In other embodiments which may be combined
with the preceding embodiments, the host cell surface molecule may
be covalently linked to the first binding, site. In other
embodiments which may be combined with the preceding embodiments,
the host cell surface molecule may be covalently linked to the
first binding site through a disulfide bond. In other embodiments
which may be combined with any of the preceding embodiments, the
host cell surface molecule may include a first agglutinin which may
be Aga1p. In other embodiments which may be combined with any of
the preceding embodiments, the host cell surface molecule may be
attached to the cell membrane via at GPI anchor. In other
embodiments which ma be combined with any of the preceding
embodiments, wherein the first binding site comprises a second
agglutinin which may be Aga2p. In other embodiments which may be
combined with any of the preceding embodiments, the second binding
site may be covalently linked to the display molecule. In other
embodiments which may be combined with any of the preceding
embodiments, the second binding site may be covalently linked to
the display molecule through disulfide bonds. In other embodiments
which may be combined with any of the preceding embodiments, the
second binding site includes a PDZ domain which may be the PDZ
domain of InaD which may have the amino acid sequence of SEQ ID NO:
8. In other embodiments which may be combined with any of the
preceding embodiments, the display molecule includes a NorpA ligand
which may be at the C-terminus which may have the amino acid
sequence of SEQ ID NO: 9. In other embodiments which may be
combined With any of the preceding embodiments except where the
second binding site includes a PDZ domain or where the display
molecule comprises a NorpA ligand, the display molecule includes a
PDZ domain which may be the PDZ domain of InaD which may have the
amino acid sequence of SEQ ID NO: 8. In other embodiments which may
be combined with ally of the preceding embodiments except where the
second binding site includes a PDZ domain or where the display
molecule comprises a NorpA ligand, the second binding site includes
a NorpA ligand which may be at the C-terminus which may have the
amino acid sequence or SEQ ID NO: 9. In other embodiments which may
be combined with any of the preceding embodiments, wherein the
modified polypeptide may be a scaffold protein, a signal
transduction protein, an antibody, an immunoglobulin, an
immunoadhesin, a receptor, a ligand an oncoprotein, a transcription
factor, or an enzyme. In other embodiments which may be combined
with any of the preceding embodiments, display molecule may be a
fibronectin polypeptide which may include an F10 polypeptide. In
other embodiments which may be combined with any of the preceding
embodiments, the display molecule includes a secretion signal
peptide may be an MFalpha, secretion signal sequence, a
glucoamylase, an Aga2 secretion signal sequence, an Flo1p secretion
signal sequence, an invertase secretion signal sequence, or an acid
phosphatase secretion signal sequence. In other embodiments which
may be combined with any of the preceding embodiments, the
secretion signal peptide may be an MFalpha/HSA hybrid leader
peptide. In other embodiments which may be combined with any of the
preceding embodiments, expression of the display molecule is under
the control of a first inducible promoter which may be an AOX 1
promoter, a Cup 1 promoter, or a Gal promotor. In other embodiments
which may be combined with any of the preceding embodiments, the
expression of the adapter molecule is under the control of a second
inducible promoter which may be an AOX 1 promoter, a Cup 1
promoter, or a Gal promotor, in other embodiments which may be
combined with any of the preceding embodiments, the host cell may
be a yeast cell which may Pichia pastoris or Saccharomyces
cerevisiae.
[0009] Another aspect includes libraries of host cells which
include least two host cells in accordance with the preceding
aspect and any and all of its embodiments where each host cell
includes a different modified polypeptide.
[0010] Yet another aspect includes methods for displaying a
modified polypeptide which includes (a) providing a host cell
comprising a cell surface molecule attached to the surface of the
cell and a first nucleic acid encoding a display polypeptide
comprising a modified polypeptide, (b) contacting the host cell
with an adapter molecule comprising a first binding site and a
second binding site under conditions wherein the first binding site
binds to the cell surface molecule, and then (c) incubating, the
host cell under conditions wherein the host cell exports the
display polypeptide outside the host cell under conditions wherein
the second binding site binds to the display polypeptide, where the
first binding site binds specifically to the cell surface molecule
and cannot bind to the display molecule, the second binding site
binds specifically to the display polypeptide and cannot hind to
the cell surface molecule, and the adapter molecule is not a
component of the modified polypeptide. In other embodiments, the
host cell may display at least 10.sup.2, at least 10.sup.3, at
least 10.sup.4, or at least 10.sup.5 modified polypeptides. In
other embodiments which may be combined with the preceding
embodiments, the host cell surface molecule may be covalently
linked to the first binding site. In other embodiments which may be
combined with the preceding embodiments, the host cell surface
molecule may be covalently linked to the first binding site through
a disulfide bond. In other embodiments which may be combined with
any of the preceding embodiments, the host cell surface molecule
may include a first agglutinin which may be Aga1p. In other
embodiments Which may be combined with any of the preceding
embodiments, the host cell surface molecule may be attached to the
cell membrane via a GPI anchor. In other embodiments which may be
combined with any of the preceding embodiments, wherein the first
binding site comprises a second agglutinin which may be Aga2p. In
other embodiments which may be combined with any of the preceding
embodiments, the second binding site may be covalently linked to
the display molecule. In other embodiments which may be combined
with any of the preceding embodiments, the second binding site may
be covalently linked to the display molecule through disulfide
bonds. In other embodiments which may be combined with any of the
preceding embodiments, the second binding site includes a PDZ
domain which may be the PDZ domain of InaD which may have the amino
acid sequence of SEQ ID NO: 8. in other embodiments which may be
combined with any of the preceding embodiments, the display
molecule includes a NorpA ligand which may heat the C-terminus
which may have the amino acid sequence of SEQ ID NO: 9. In other
embodiments which may be combined with any of the preceding
embodiments except where the second binding site includes a PDZ
domain or where the display molecule comprises a NorpA ligand, the
display molecule includes a PDL domain which may be the PDZ domain
of InaD which may have the amino acid sequence of SEQ ID NO: 8. In
other embodiments which may be combined with any of the preceding
embodiments except where the second binding site includes a PDZ
domain or where the display molecule comprises a NorpA ligand, the
second binding site includes a NorpA ligand which may be at the
C-terminus which may have the amino acid sequence of SEQ ID NO: 9.
In other embodiments which may be combined with any of the
preceding embodiments, wherein the modified polypeptide may be a
scaffold protein, a signal transduction protein, an antibody, an
immunoglobulin, an immunoadhesin, a receptor, a ligand, an
oncoprotein, a transcription factor, or an enzyme. In other
embodiments which may be combined with any of the preceding
embodiments, display molecule may be a fibronectin polypeptide
which may include an F10 polypeptide. In other embodiments which
may be combined with any of the preceding embodiments, the display
molecule includes a secretion signal peptide may be an MFalpha
secretion signal sequence, a glucoamylase an Aga2 secretion signal
sequence, an Flo1p secretion signal sequence, an invertase
secretion signal sequence, or an acid phosphatase secretion signal
sequence. In other embodiments which may be combined with any of
the preceding embodiments, the secretion signal peptide may be an
MFalpha/HSA hybrid leader peptide. In other embodiments which may
be combined with any of the preceding embodiments, expression of
the display molecule is under the control of a first inducible
promoter which may be an AOX 1 promoter,a Cup 1 promoter, or a Gal
promotor. In other embodiments which may be combined with any of
the preceding embodiments, the expression of the adapter molecule
is under the control of a second inducible promoter which may be an
AOX 1 promoter, a Cup 1 promoter, or a Gal promotor. In other
embodiments which may be combined with any of the preceding
embodiments, the host cell may be a yeast cell winch may be Pichia
pastoris or Saccharomyces cerevisiae.
[0011] Still another aspect includes methods for generating a host
cell display library which includes introducing into a plurality of
host cells a display library of first nucleic acids each encoding a
display polypeptide comprising a modified polypeptide, wherein at
least two of the introduced first nucleic acids encode different
modified polypeptides, wherein each host cell comprises a second
nucleic acid which encodes a cell surface polypeptide and a third
nucleic acid which encodes an adapter molecule comprising a first
binding site and a second binding site where the first binding site
binds to the cell surface molecule but not the display polypeptide,
the second binding site binds to the display polypeptide but not
the cell surface molecule, and the adapter molecule is not as
component of the modified polypeptide. This aspect may be combined
with any of the embodiments of the preceding aspects.
[0012] The foregoing are non-limiting examples of the present
invention. Additions aspects and embodiments may be found
throughout the specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a schematic of the three component system
including the cell surface molecule, the adapter molecule including
the first and second binding sites and the display molecule with
the binding partner for the second binding site (a binding
polypeptide in this embodiment) and a modified polypeptide as the
molecule being displayed. In this embodiment, the host cell is a
yeast cell.
[0014] FIG. 2 shows the pPlC3.5 AGA1 vector for yeast expression of
the Aga1p as the cell surface molecule.
[0015] FIG. 3 shows the pPlC6 A AGA2-InaD vector for yeast
expression of the Aga2p-InaD fusion polypeptide as the adapter
molecule.
[0016] FIG. 4 shows the pPlCHOLl-1 MFalpha1 Hsa-Fn10-NorpA vector
for yeast expression of the MFalpha1Hsa-Fn10-NorpA fusion
polypeptide as the display molecule where the NorpA is the binding
partner of the second binding site of the adapter molecule (i.e.,
InaD) and the fibronectin F10 domain is modified polypeptide.
[0017] FIG. 5 shows the pPlCHOL1-C MFalpha1 Hsa-Fn10-NorpA vector
for yeast expression of the MFalpha1Hsa-Fn10-NorpA fusion
polypeptide as the display molecule where the NorpA is the binding
partner of the second binding site of the adapter molecule (i.e.,
InaD) and the fibronectin HO domain is modified polypeptide.
[0018] FIG. 6 shows the pYD NBC1 Aga2-InaD vector for yeast
expression of the Aga2p-InaD fusion polypeptide as the adapter
molecule.
[0019] FIG. 7 shows the pYS HSA MFalpha1 Fn10 NorpA vector for
yeast expression of the MFalpha1HSA-Fn10-NorpA fusion polypeptide a
the display molecule where the NorpA is the binding partner of the
second binding site of the adapter molecule (i.e., InaD) and the
fibronectin F10 domain is modified polypeptide.
[0020] FIG. 8 shows the pYS MFalpha1 HSA NorpA vector for yeast
expression of the MFalpha1-HSA-NorpA fusion polypeptide as the
display molecule where the NorpA is the binding partner of the
second binding site of the adapter molecule (i.e., InaD) and the
HSA is the displayed polypeptide.
[0021] FIG. 9A-E are FACS analysis graphs showing protein surface
expression of yeast cells expressing either fibronectin (pYS6/CT
HSA MFalpha1 Fn10 NorpA) or HSA (pYS6/CT MFalpha1-HSA-NorpA) in
which the yeast cells are stained with anti-myc antibody and APC
labeled secondary anti-mouse antibody. FIG. 9A is the a control
with unstained yeast cells: FIG. 9B are uninduced yeast cells
expressing fibronectin; FIG. 9C are induced yeast cells expressing
fibronectin showing a shift in the curve; FIG. 9D are uninduced
yeast cells expressing HSA; and FIG. 9E are induced yeast cells
expressing HSA showing a shift in the curve.
[0022] FIG. 10A-E are images of FMAT analysis of yeast cells
expressing either fibronectin (pYS6/CT HSA MFalpha1 Fn 10 NorpA) or
HSA (pYS6/CT HSA-NorpA) in which the yeast cells are stained with
anti-myc mouse monoclonal antibody and APC labeled secondary
anti-mouse antibody and subjected to FMAT confocal fluorescence.
FIG. 10A is the a control with unstained yeast cells: FIG. 10B are
uninduced yeast cells expressing fibronectin; FIG. 10C are induced
yeast cells expressing fibronectin appearing as white spots; FIG.
101) are uninduced yeast cells expressing HSA; and FIG. 10E are
induced yeast cells expressing HSA appearing, as white spots.
[0023] FIG. 11 shows the pYS MFalpha1 scFv lysozyme NorpA vector
for yeast expression attic MFalpha1 scFv lysozyme NorpA fusion
polypeptide as the display molecule where the NorpA is the binding
partner of the second binding site of the adapter molecule (i.e.,
InaD) and the lysozyme is modified polypeptide.
[0024] FIG. 12 shows a reversed system in which the pYD NBC1
Aga2-NorpA vector is used for yeast expression of the NBC1
Aga2-NorpA,
[0025] FIG. 13 shows a reversed system in which the
pYS6/CT*MFalpha1-InaD-Fn10 vector is used for yeast expression of
the MFalpha1-InaD-Fn10.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] As used herein the term "host cell" refers to a eukaryotic
cell that has been modified to express a cell surface molecule, an
adapter molecule, and a display molecule. Furthermore, it should be
understood that the host cell secretes or excretes the display
molecule prior to binding the display molecule to the adapter
molecule on the surface of the host cell.
[0027] As used herein the term "cell surface molecule" refers to a
peptide, polypeptide, binding domain, ligand, lipid, or
carbohydrate that is directed to the extracellular surface of the
host cell. The cell surface molecule may be anchored to the cell
surface by covalent binding or non-covalent binding. The cell
surface molecule may include a phospholipid, carbohydrate or
protein through which it attaches to the surface of the host cell.
The cell surface molecule may be a polypeptide that binds to, or is
conjugated to a phospholipid, carbohydrate, or a polypeptide on the
surface of the cell. For example, the polypeptide may use a
phosphatidyl-inositol-glycan (GPI) anchor to attach to the surface
of the host cell, such as .alpha.-agglutinins, .alpha.-agglutinins,
and flocculins. The cell surface molecule may also be a
transmembrane protein with a binding domain located on the surface
of the host cell that can bind to the first binding site of the
adapter molecule.
[0028] As used herein the term "adapter molecule" refers to a
peptide, polypeptide, binding domain, ligand, lipid, or
carbohydrate or combination of the foregoing that has two distinct
binding sites. The adapter molecule has a binding site that
specifically binds the cell surface molecule and a second distinct
binding she that specifically binds the display molecule. Without
limiting the invention, the two binding sites of the polypeptide ma
bi polypeptide domains each with its on binding affinity to a
different molecule that are fused together. For example, the
polypeptide may be an a-agglutinin subunit, such as Aga2p, fused to
a PDZ domain, or it may be a flocculin, such as Flo1, fused to a
PDZ domain.
[0029] As used herein the term "first binding site" refers to a
region of the adapter molecule that specifically recognizes and
binds at least a portion of the cell surface molecule. For example,
the first binding site may comprise a peptide, polypeptide, binding
domain, ligand, lipid, or carbohydrate or combination thereof that
specifically binds to a cell surface molecule which could include,
without limitation, a peptide, binding domain, ligand, protein,
lipoprotein, lipid, or carbohydrate. More specifically, but without
limiting the invention, the first binding, site may refer to the
Aga2p subunit of .alpha.-agglutinin that specifically binds to the
Aga1p subunit of .alpha.-agglutinin through disulfide bonds. In
general, any to molecular binding partners may be used for the
first binding site and the corresponding portion of the cell
surface molecule. Examples include Ni.sup.2+ ions and polyhistidine
tags, sugar residues and Concanavalin A,
p-aminophenyl-.beta.-D-thiogalactoside and .beta.-galactosidase
(Germino et al., Proc. Natl. Acad. Sci. USA 80 6848 (1983),
glutathione and glutathione-S-transferase (Smith, D. B. and
Johnson, K. S. Gene 67:311 (1988)); staphylococcal protein A and
IgG (Uhlen, M. et al. Gene 23:369 (1983)), calmodulin nickel
binding proteins (CNBP) and calmodulin agarose Stofko-Hahn R. E. et
al. FEBS Lett. 302(3):274-278); streptavidin or avidin and biotin
(Takashige, S. and Dale, G. L., Proc. Natl. Acad. Sci. USA.
85:1647-1651 (1988)); amylase and maltose-binding protein domain
from the malE gene of E. coli (Bach, H. et al, J. Mol. Biol.
312:79-93 (2001)), any epitope and its corresponding antibody (Sec,
Kolodziej. P. A. and Young., R. A Methods Enzymol. 194:508-519
(1991), e.g., the FLAG.TM. octapeptide or antidigoxygenin antibody
and digoxygenin).
[0030] As used herein the term "second binding site" refers to a
region of the adapter molecule that specifically recognizes and
hinds the display molecule. For example, the second binding site
may comprise a peptide, polypeptide, binding domain, ligand, lipid,
or carbohydrate that specifically binds to a peptide, ligand,
protein, lipoprotein, lipid, or carbohydrate comprising the display
molecule. More specifically, but without limitations, the second
binding site may refer to a PMZ domain that specifically binds to a
NorpA ligand. Any of the binding pairs suitable for the first
binding site may also be used for the second binding site so long
as they do not recognize the same partners.
[0031] As used herein the term "display molecule" refers to a
molecule that can be localized to the surface of the host cell via
binding of the adapter molecule on the surface of the host cell.
The display molecule will typically comprise the molecule (or
library of molecules) to be displayed and a binding partner that is
specifically bound by the second binding site of the adapter
molecule. In certain instances the molecule to be displayed and the
binding partner may be one in the same. By way of example, the
display molecule may comprise a peptide, polypeptide, binding
domain, ligand, lipid, or carbohydrate or combination thereof. It
should be understood that the display molecule is expressed or
otherwise generated within the host cell and is secreted or
excreted out of the cell so as to be displayed on the surface of
said cell. The display molecule may comprise a library of varied
molecules that can be screened for binding to a target or for
improved or altered activity. In certain embodiments, the library
may comprise modified polypeptides. The display molecule may also
comprise a tag or peptide that can be labeled so as to detect
binding of the display molecule to the cell surface, or sort host
cells displaying said molecule.
[0032] As used herein the term "modified polypeptide" refers to any
polypeptide of interest that is fused to a peptide, polypeptide,
binding domain, ligand, lipid, or carbohydrate that specifically
binds to a peptide, ligand, protein, lipoprotein, lipid, or
carbohydrate comprising the second binding site of the adapter
molecule, and is displayed on the surface of the host cell (and is
therefore a component of the display molecule). Non-limiting
examples of the modified polypeptide are scaffold proteins, signal
transduction proteins, antibodies, immunoglobulins, immunoadhesins,
receptors, ligands, oncoproteins, transcription factors, and
enzymes,
[0033] As used herein, the term "plurality of display molecules"
refers to at least two copies of the display molecule displayed on
the surface of host cells. In certain instances, each unique
display molecule is displayed by a different host cell.
[0034] As used herein the term "component of the modified
polypeptide" refers to any naturally occurring binding partners of
any fragment of the modified peptide. Non-limiting examples include
an immunoglobulin light chain binding to an immunoglobulin heavy, a
biotin molecule binding avidin, two subunits of .alpha.-hemoglobin
dimerizing, a myosin heavy chain binding to a myosin light chain,
two monomers of glycophorin. A dimerizing, or two monomers of any
naturally occurring dimer protein binding to one another.
[0035] As used herein the term "library of host cells" refers to a
plurality of host cells, wherein each host cell comprises a
non-identical modified polypeptide that is displayed on the surface
of the cell.
[0036] As used herein the term "non-identical modified polypeptide"
refers to the amino acid sequence of at least two modified
polypeptides, wherein each amino acid sequence comprises amino acid
substitutions, insertions, or deletions which differentiate one
modified polypeptide displayed on the surface of a host cell from
another modified polypeptide displayed on the surface of a second
host cell.
[0037] As used herein the term "Fn10" refers to the tenth type III
domain of human fibronectin.
[0038] Display Molecules
[0039] The display molecules may be used to display any molecule
that may be expressed or otherwise generated in a host cell. Non
limiting examples of such molecules follow.
[0040] Antibody Scaffold
[0041] The display molecules may be immunoglobulins. Methods of
generating libraries of immunoglobulins were initially developed to
display the immunoglobulins via phage, but additional methods of
display have since been developed. All of the methods of generating
libraries for these alternative methods of display may be adapted
to allow display using the methods disclosed herein. Exemplary
method of generating libraries of antibodies may be found in U.S.
Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.:
U.S. Pat. No 5,427,908 and 5,580,717 to Dower et al.: U.S. Pat.
Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat.
Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and
6,593,081 to Griffiths et al.
[0042] Non-antibody Scaffold
[0043] Known non-immunoglobulin frameworks or scaffolds which may
be displayed using the methods disclosed herein include, but are
not limited to, fibronectins (Compound Therapeutics, Inc., Waltham,
Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),
domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv
(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,
Freising, Germany), small modular immuno-pharmaceuticals (Trubion
Pharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.
(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and
affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle,
(Germany), protein epitope munches (Polyphor Ltd, Allschwil,
Switzerland).
[0044] (i):Fibronetins
[0045] The adnectin scaffolds are based on fibronectin type III
domain (e.g., the tenth module of the fibronectin type III (10 Fn3
domain). The fibronectin type III domain has 7 or 8 beta strands
which are distributed between two beta sheets, which themselves
pack against each other to form the core of the protein, and
further containing loops (analogous to CDRs) which connect the beta
strands to each other and are solvent exposed. There are at least
three such loops at each edge of the beta sheet sandwich, where the
edge is the boundary of the protein perpendicular to the direction
of the beta strands. (U.S. Pat. No. 6,673,901).
[0046] These fibronectin-based scaffolds are not an immunoglobulin,
although the overall fold is closely related to that of the
smallest functional antibody fragment, the variable region of the
heavy chain, which comprises the entire antigen recognition unit in
camel and llama IgG. Because of this structure, the
non-immunoglobulin fibronectin molecule mimics antigen binding
properties that are similar in nature and affinity to those of
antibodies. These scaffolds can be used in a loop randomization and
shuffling strategy in vitro that is similar to the process of
affinity maturation of antibodies in vivo. These fibronectin-based
molecules can be used as scaffolds where the loop regions of the
molecule can be replaced with CDRs of the disclosure using standard
cloning techniques.
[0047] (ii) Ankyrin--Molecular Partners
[0048] The technology is based on using proteins with ankyrin
derived repeat modules as scaffolds for bearing variable regions
which can be used for binding to different targets. The ankyrin
repeat module is a 33 amino acid polypeptide consisting of two
anti-parallel .alpha.-helices and a .beta.-turn. Binding of the
variable regions is mostly optimized by using ribosome display.
[0049] (iii) Maxybodies/Avimers--Avidia
[0050] Avimers are derived from natural A-domain containing protein
such as LRP-1. These domains are used by nature for protein-protein
interactions and in human over 250 proteins art structurally based
on A-domains. Avimers consist of a number of different "A-domain"
monomers (2-10) linked via amino acid linkers. Avimers can be
created that can bind to the target antigen using the methodology
described in, for example, 20040175756; 20050053973; 20050048512;
and 20060008844.
[0051] (vi) Protein A--Affibody
[0052] Affibody.RTM. affinity ligands are small, simple proteins
composed of a three-helix bundle based on the scaffold of one of
the IgG-binding domains of Protein A. Protein A is a surface
protein from the bacterium Staphylococcus aureus. This scaffold
domain consists of 58 amino acids, 13 of which are randomized to
generate Affibody.RTM. libraries with a large number of ligand
variants (See e.g., U.S. Pat. No. 5,831,012). Affibody.RTM.
molecules mimic antibodies, they have a molecular weight of 6 kDa,
compared to the molecular weight of antibodies, which is 150 kDa.
In spite of its small size, the binding site of Affibody molecules
is similar to that of an antibody.
[0053] (v) Anticalins--Pieris
[0054] Anticalins.RTM. are products developed by the company Pieris
ProteoLab AG. They are derived from lipocalins, a widespread group
of small and robust proteins that are usually involved in the
physiological transport or storage of chemically sensitive or
insoluble compounds. Several natural lipocalins occur in human
tissues or body liquids.
[0055] The protein architecture is reminiscent of immunoglobulins,
with hypervariable loops on top of a rigid framework. However, in
contrast with antibodies or their recombinant fragments, lipocalins
are composed of a single polypeptide chain with 160 to 180 amino
acid residues, being just marginally bigger than a single
immunoglobulin domain.
[0056] The set of four loops, which makes up the binding pocket,
shows pronounced structural plasticity and tolerates a variety of
side chains. The binding site can thus be reshaped in a proprietary
process in order to recognize prescribed target molecules of
different shape with high affinity and specificity.
[0057] One protein of lipocalin family, the bilin-binding protein
(BBP) of Pieris Brassicae has been used to develop anticalins by
mutagenizing the set of four loops. One example of a patent
application describing "anticalins" is PCT WO199916873.
[0058] (vi) Affilin--Scil Proteins
[0059] Affilin.TM. molecules are small non-immunoglobulin proteins
which are designed for specific affinities towards proteins and
small molecules. New Affilin.TM. molecules can be very quickly
selected from two libraries, each of which is based on a different
human derived scaffold protein.
[0060] Affilin.TM. molecules do not show any structural homology to
immunoglobulin proteins. Scil Proteins employs two Affilin.TM.
scaffolds, one of which is gamma crystalline, a human structural
eye lens protein and the other is "ubiquitin" superfamily proteins.
Both human scaffolds are very small, show high temperature
stability and are almost resistant to pH changes and denaturing
agents. This high stability is mainly due to the expanded beta
sheet structure of the proteins. Examples of gamma crystalline
derived proteins are described in WO200104144 and examples of
"ubiquitin-like" proteins are described in WO2004106368.
[0061] (vii) Protein Epitope Mimetics (PEM)
[0062] PEM are medium-sized, cyclic, peptide-like molecules (MW
1-2kDa) mimicking beta-hairpin secondary structures of proteins,
the major secondary structure involved in protein-protein
interactions.
[0063] Non-scaffold
[0064] In addition to scaffolds which are useful for de novo
generation of molecule with specific affinity, the methods
disclosed herein may be used to display any other biological
molecule that can be expressed or otherwise generated in the host
cell. Libraries of such biological molecules, particularly
polypeptides which can be encoded by polynucleotides for easy
expression by the host cell, may be screened for improved
characteristics of interest such as improved binding between
receptor and ligand where the receptor or the ligand are part of
the display molecule or improved enzymatic activity where the
enzyme is part of the display molecule.
[0065] Expression Systems
[0066] Expression vectors may be used to express one or more of the
cell surface molecules, the adapter molecule and the display
molecule, in the host cell. Expression vectors for eukaryotic host
cells typically include (i) eukaryotic DNA elements that control
initiation of transcription, such as a promoter, (ii) eukaryotic
DNA elements that control the processing of transcripts, such as a
transcription termination/polyadenylation signal sequence, and
(iii)optionally eukaryotic DNA elements that control replication in
the eukaryotic host cell if the vector is to be independently
replicated (e.g., non-integrating vectors). To ease construction of
such expression vectors, the vectors may optionally include (iv)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector when manipulating the vector in
the bacterial host cell. Appropriate eukaryotic expression vectors
for use with fungal, yeast, and mammalian cellular hosts are known
in the art, and are described in, for example, Powell et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
[0067] Yeast host cells are of particular interest and include
Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
These vectors include YIp-based vectors, such as YIp5, YRp vectors,
such as YRp17, YEp vectors such as YEp13 and YCp vectors, such as
YCp19. A number of vectors exist for the expression of recombinant
proteins in yeast, Other example of the YEp vectors include YEp24,
YEp51, and YEp52,which are cloning and expression vehicles useful
in the introduction of genetic constructs into S. cerevisiae (see,
e.g., Broach et al. (1983) in Experimental Manipulation of Cleric
Expression, ed. M. Inouye Academic Press, p 83), These vectors are
also shuttle vectors in that they can replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the
replication determinant of the yeast 2 micron plasmid.
[0068] Suitable promoters for function in yeast include the
promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes
(Hess et al., J. Adv. Enzyme Req. 7, 149 (1968); and Holland et al,
Biochemistry 17, 4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phospho-glucose isomerase, and glucokinase. Suitable
vectors and promoters for use in yeast expression are further
described in R. Hitzeman et al., EP073,657. Other suitable
promoters for expression in yeast include the promoters from GAL1
(galactose), PGK (phosphoglycerate kinase), ADH (alcohol
dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol
dehydrogenase), and the like. Many yeast cloning vectors readily
available and can be modified following the above discussion. Still
other promoters, which have the additional advantage of
transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the afore-mentioned metallothionein and
glycetaldehyde-3-phosphate dehydrogenase, as well as enzymes
responsible for maltose and galactose utilization. Finally,
promoters that are active in only one of the two haploid mating
types may be appropriate in certain circumstances. Among these
haploid-specific promoters, the pheromone promoters MFa1 and
MF.alpha.1 are of particular interest.
[0069] Secretion from yeast host cells of the components including
the adapter molecule (if produced in the host cell) and the display
molecule may be increased by use any available secretion signal
sequences of yeast proteins, One example is the leader sequence of
a precursor of yeast mating pheromone, .alpha.-factor, which has
also been used to direct secretion of heterologous proteins in
yeast (See. e.g., Valenzuela, P., eds pp. 269-280, Butterworths,
London; Brake, A. J. (1990) Meth. Enzymol. 185, 408-441). The
.alpha.-factor leader sequence, in addition to the N-terminal
signal peptide of 17 residues, includes a hydrophilic pro-region
which contains 72 residues and bears three sites of N-linked
glycosylation. The pro-region is extensively glycosylated in the ER
and Golgi and is cleaved by Kex2 endopeptidase in the late Golgi
compartment. The presence of the pro-region at the N-terminus is
believed to allow some heterologous proteins to pass the quality
control in the ER and to reach the periplasm.
[0070] Another example is the leader sequence from yeast invertase
(MLLQAFLFLLAGFAAKISADAHKS) (SEQ ID NO: 1). This leader sequence has
been demonstrated to be cleaved from nascent heterologous peptide
upon entrance into the endoplasmic reticulum. The enzyme
responsible for cleavage of the pre sequence, Kex2, resides in the
trans Golgi. A further example is the signal sequence of yeast acid
phosphatase which may be used to direct the secretion of the
components disclosed herein.
[0071] Methods for transforming S. cerevisiae cells with exogenous
DNA and producing recombinant polypeptides therefrom are disclosed
by, for example, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et
al., U.S. Pat. No. 4,931,373, Brake, U.S. Pat. No. 4,870,008, Welch
et al, U.S. Pat. No. 5,037,743, and Murray et at U.S. Pat. No.
4,845,075. Transformed cells are selected by phenotype determined
by the selectable, marker, commonly drug resistance or the ability
to grow in the absence of a particular nutrient (e.g., leucine). A
preferred vector system for use in Saccharomyces cerevisiae is the
POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No.
4,931,373), which allows transformed cells to be selected by growth
in glucose containing media.
[0072] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al,. J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Laambowitz, U.S. Pat. No. 4,486,533.
[0073] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808 Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica it is preferred that the promoter and
terminator in the plasmid be that of a P. methanolica gene, such as
a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other
useful promoters include those of the dihydroxyacetone synthase
(DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. For large-scale,
industrial processes where it is desirable to minimize the use of
methanol, it is preferred to use host cells in which both methanol
utilization genes (AUG1 and AUG2) are deleted, For production of
secreted proteins host cells deficient in vacuolar protease genes
(PEP4 and PRB1) are preferred. Electroporation is used to
facilitate the introduction of a plasmid containing DNA encoding a
polypeptide of interest into P. methanolica cells. P. methanolica
cells can be transformed by electroporation using an exponentially
decaying, pulsed electric field having a field strength of from 2.5
to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t)
of from 1 to 40 milliseconds, most preferably about 20
milliseconds.
[0074] For use of mammalian host cells, mammalian expression
vectors are also well known in the art and may be used as well.
Examples of suitable mammalian host cells include African green
monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney
cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells
(BHK-21,BHK-570; ATCC CRL8544, ATCC CRL 10314), canine kidney cells
(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC
(CCL61; CHO DG44 (Chasin et al., Som, Cell. Molec. Genet. 12:555,
1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC,
CCL2.2), rat hepatoma cells (H4-II-E, ATCC CRL 1548)
SV40-transformed monkey kidney cells (COS-1 ATCC CRL 1650) and
murine embryonic cells (NIH-3T3; ATCC CRL 1658).
EXAMPLES
[0075] The following provides non-limiting examples of the systems,
compositions and methods disclosed herein. Proteins can be
displayed on the surface of yeast cells by utilizing the PDZ domain
of the yeast InaD protein and the C-terminal 5 amino acids of the
yeast NorpA protein. This three component protein display system
consists of a vector expressing the protein to be displayed with a
secretion signal fused at its N-terminus, and the NorpA ligand
fused at the C-terminus; a second vector expressing an adapter
protein that can bind specifically to a yeast cell wall protein,
and which is fused to the PDZ domain of InaD, which binds
specifically to the NorpA ligand; and a third vector that express a
yeast cell wall protein that binds specifically to the adapter
protein. This system has been adapted for use in Sacchromyces
cerrevisiae and Pichia pastoris.
Example 1
Yeast Display Using InaD/NorpA Interaction in Pichia pastoris
[0076] The protein display system for P. pastoris was developed to
display a fibronectin type 111 domain (Fn10), by fusing a hybrid
secretion sequence (MFalpha/HSA) or a yeast leader sequence
(MFalpha1) at N-terminus of Fn10 and fusing the NorpA ligand to its
C-terminus. Once expressed, the Fn10 was secreted from the cell and
the NorpA ligand bound specifically to the PDZ domain of InaD
through disulfide bonds. The InaD was fused to the C-terminus of
the Agap2 protein. The Aga2p-InaD fusion protein served as the
adapter protein, and the N-terminal Aga2p bound Aga1p, which was
immobilized on the surface of the cell. Aga2p bound specifically to
Aga1p through disulfide bonds.
[0077] The three component system consisting of the Fn10-NorpA
fusion protein, the
[0078] Aga2p-InaD fusion protein, and the Aga1p cell surface
protein were cloned into pPIC expression vectors, under the control
of an inducible promoter. The inducible promoter used was the AOX1
promoter, which is induced by methanol. Thus when methanol was
added to yeast cells transformed with the vectors, the three
proteins were expressed. Aga1p as expressed on the surface of the
cell, Aga2p-InaD was localized to the cell surface where the
N-terminal region of Aga2p-InaD bound to Aga1p. Fn10-NorpA was
localized to the secretory pathway, was secreted from the cell, and
bound InaD via the C-terminal NorpA ligand (FIG. 1). The system can
also be switched such that the InaD is fused to Aga1 and NorpA is
fused to Aga2p (See FIGS. 12 and 13).
[0079] A c-myc epitope to was fused between the NorpA ligand and
the C-terminus of Fn10. The c-myc epitope allowed for the detection
of the displayed fibronectin by using a c-myc antibody. The
fluorescently labeled c-myc antibody bound to the c-myc epitope on
the surface of the cell was detected by fluorescence-activated cell
sorting (FA(S).
[0080] Strains and Media
[0081] The Escherichia coli Top10 strain (Invitrogen Carlsbad,
Calif.) was used as the host strain for recombinant DNA
manipulation. The P. pastoris GS115 strain (Invitrogen Co.,
Carlsbad, Calif.) was used for the production of the fusion protein
AGA2-InaD and HSA/MFalpha1-Fn10-NorpA. E. coli as cultivated in LB
medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride)
containing 100 ug/mL ampicillin. P. pastoris was cultivated in BMGY
medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate
buffer (pH 6.0), 1.34% yeast nitrogen base, 4.times.10.sup.-5%
biotin, and 1% glycerol), and BMMY medium (1% yeast extract, 2%
peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% yeast
nitrogen base, 4.times.10.sup.-5% biotin, and 0.5-2.0%
methanol).
[0082] Construction of Expression Plasmids
[0083] The gene corresponding to AGA1 as synthesized by Geneart and
subcloned into pPlC3.5 (Invitrogen). The resulting vector was named
pPlC3.5-AGA1 (FIG. 2). The AGA2-InaD anchor gene was synthesized by
Geneart (Germany) and subcloned into expression vector pPIC6a
(Invitrogen) using Bst1 and EcoR1 restriction sites. The resulting
vector was named pPlC6-AGA2-InaD (FIG. 4). The fibronectin
construct consists of the MFalpha1/HSA hybrid leader followed by
the fibronectin fused at the C-terminus to the NorpA ligand
sequence. The complete gene was synthesized by Geneart (Germany)
and subcloned into pPlCHOLl-1 (Mobitec). The resulting vector was
named pPlCHOLl-1 MFalpha1Hsa-Fn10-NorpA (FIG. 6).
[0084] Yeast Transformation.
[0085] Electro-competent P. pastoris GS115 (Invitrogen) strain was
prepared according to the protocol specified by the supplier and
co-transformed with SalI-digested pPlC3.5-AGA1, pPlC6-AGA2-InaD.
and pPlCHOLl-1 MFalpha1Hsa-Fn10-NorpA.
[0086] Cultivation Conditions
[0087] The yeast transformants were precultivated in BMGY medium
containing 100 ug/ml Zeocin and 200 ug/ml Blasticidin at 30.degree.
C. for 16 hr, and used to inoculate 200 ml of BMGY medium
(containing 100 ug/ml Zeocin and 200 ug/ml Balsticidin) in a 1 l
baffle flask to give an initial OD.sub.600 value of 0.1. After 24
hr. of cultivation, the culture was centrifuged at 1000 g for 10
min, and resuspended in BMMY medium (+100 ug/ml Zeocin and 200
ug/ml Blasticidin) containing 0.5%, 1.0%, or 2.0% methanol. To
maintain the induction of the fusion proteins, 100% methanol was
added every 24 hr. to the culture to the final concentrations
mentioned above. Analysis of displayed fibronectins on the surface
of yeast is performed using FACS and anti-myc antibody.
Example 2
Switch System to Secrete or Display Fibronectins on the Surface of
Pichia pastoris
[0088] One variant of the above display system enables the choice
between secretion and display of proteins from P. pastoris. To
achieve this, the fibronectin construct consisting of the
MFalpha1/HSA hybrid leader followed by fibronectin fused at the
C-terminus to the NorpA ligand sequence is cloned into pPlCHOLl-C
instead of pPlCHOLl-1. The resulting vector is named pPlCHOLl-C
Mfalpha1Hsa-Fn10-NorpA (FIG. 8). The key difference between the two
vectors is the promoter, which in pPlCHOLl-1 is the AOX1 promoter
induced by methanol, and in the pPlCHOLl-C is the Cup1 promoter
induced by copper. To display the fibronectin on the surface of P.
pastoris, AGA1 and AGA2-InaD are induced with methanol, while
pPlCHOL1-C is induced with copper. This allows for the capture of
the secreted fibronectin on the surface of yeast mediated through
the tight InaD/NorpA interaction. For secretion of the fibronectin
without displaying the protein on the surface of yeast, induction
with copper is sufficient. Without the induction of AGA1 and
AGA2-InaD (driven by methanol) the binding partner for NorpA
(AGA1/AGA2-InaD) is not present on the surface of yeast and
therefore the fibronectin will be secreted.
Example 3
Yeast Display Using InaD/NorpA Interaction in Saccharomyces
cerevisiae
[0089] This example describes using the InaD/NorpA system with
other yeast strains such as Saccharomyces cerevisiae
[0090] Strains and Media
[0091] Escherichia coil Top10 (Invitrogen, Carlsbad, Calif.) was
used as the host strain for recombinant DNA manipulation. The S.
cerevisiae strain EBY100 (Invitrogen Co., Carlsbad, Calif.) was
used for the production of the fusion proteins AGA2-InaD and
MSalpha1/HSA-Fn10-NorpA or pYS6CT*MFalpha1-HSA-NorpA. E. coli was
cultivated LB medium (1% tryptone, 0.5% yeast extract, and 0.5%
sodium chloride) containing 100 u/g/mL ampicillin or 100 ug/ml
Blasticidin. EBY 100 was cultivated in CM medium-URA.
[0092] Construction of Expression Plasmids
[0093] The InaD anchor gene was synthesized by Geneart (Germany)
and subcloned in frame with the AGA2 anchor protein into the
expression vector pYD NBC1 (derivative of pYD1Invitrogen) using
HindIII and EcoRI restriction sites. The resulting vector was named
pYD_NBC1 AGA2-InaD (FIG. 10). The fibronectin construct consists of
the MFalpha1/HSA hybrid leader sequence followed by the fibronectin
fused at its C-terminus to the NorpA ligand. The complete gene was
synthesized by Geneart (Germany) and subcloned into pYS6CT
(Invitrogen)), in which the origin of replication had been replaced
by the CEN6/ARS4 region. The resulting vector was named
pYS6CT_HSA_MFalpha.sub.1_Fn10_NorpA (FIG. 12).
[0094] Plasmids were isolated from E. coli and the sequence
confirmed. The purified plasmids were then co-transformed into EBY
100 and plated out on selective media consisting of CM-TRP, +200
ug/ml Blasticidin. Transformed colonies appeared within 2 days and
were tested for display of Fibronectin by FACS analysis using an
anti-myc antibody (ccccc).
Example 4
Yeast Display Using Flo1-InaD/NorpA in Pichia pastoris
[0095] This example describes the use of an alternative expression
system, Flo1, which is used with InaD/NorpA in Pichia pastoris.
[0096] Strains and Media
[0097] The Escherichia coil Top10 strain (Invitrogen Carlsbad,
Calif.) is used as the host strain for recombinant DNA
manipulation. The P. pastoris GS115 strain (Invitrogen Co.,
Carlsbad, Calif.) is used for the production of the fusion protein
Flo1-InaD and HSA/MFalpha1-Fn10-NorpA. E. coli was cultivated in LB
medium (1% tryptone, 0.5% yeast extract, and 0.5% sodium chloride)
containing 100 ug/mL ampicillin. P. pastoris was cultivated in BMGY
medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate
buffer (pH 6.0), 1.34% yeast nitrogen base, 4.times.10.sup.-5%
biotin, and 1% glycerol), and BMMY medium (1% yeast extract, 2%
peptone, 100 mM potassium phosphate buffer (pH 6.0), 1.34% yeast
nitrogen base, 4.times.10.sup.-5% biotin, and 0.5-2.0%
methanol).
[0098] Construction of Expression Plasmids
[0099] The gene for Flo1, fused at the C-terminus to the PDZ domain
of InaD, is synthesized by Geneart (Germany) and cloned into
pPlC3.5 (Invitrogen) using a 5' EcoR1 site and a 3'Notl site. The
resulting plasmid is named pPlC3.5-Flo1-InaD. Expression of the
fused protein is driven by the methanol inducible promoter AOX1.
The fibronectin construct consists of the MFalpha1/HSA hybrid
leader followed by the fibronectin fused at the C-terminus to the
NorpA ligand sequence. The complete gene is synthesized by Geneart
(German/) and subcloned into pPlCHOLl-1 (Mobitec). The resulting
vector is named pP1CHOL1-1 MFalpha1Hsa-Fn10-NorpA. Expression of
the fibronectin construct is driven by the methanol inducible
promoter AOX1.
[0100] Yeast Transformation.
[0101] Electro-competent P. pastoris GS115 (Invitrogen) strain is
prepared according to the protocol specified by the supplier and
co-transformed with SalI-digested pPIC3.5-Flo1-InaD, and pPlCHOLl-1
MFalpha1 Hsa-Fn10-NorpA,
[0102] Cultivation Conditions
[0103] The yeast transformants are precultivated in BMGY medium
containing 100 ug/ml Zeocin and 200 ug/ml Blasticidin at 30.degree.
C. for 16 hr. and used to inoculate 200 ml of BMGY medium
(containing 100 ug/ml Zeocin and 200 ug/ml Balsticidin) in a 1 l
baffle flask to give an initial OD.sub.600 value of 0.1. After 24
hr. of cultivation, the culture is centrifuged at 1000 g for 10
min. and resuspended in BMMY medium (+100 ug/ml Zeocin and 200
ug/ml Blasticidin) containing 0.5%, 1 or 2.0% methanol. To maintain
the induction of the fusion proteins. 100% methanol is added every
24 hr. to the culture to the final concentrations mentioned above.
Analysis of displayed fibronectins on the surface of yeast is
performed using FACS and anti-myc antibody.
Example 5
Screening of a Fibronectin Library
[0104] Fibronectin Library Display
[0105] A fibronectin library is generated by methods well known in
the art, including the method disclosed in U.S. Pat. No. 6,673,901.
Other methods, such as use of error prone PCR, use of random
priming techniques, or use of computational techniques are well
known in the art and can also be used. The fibronectin library is
designed with appropriate restriction enzyme cleavage sites in
order to clone the library into yeast expression vectors.
[0106] The fibronectin library is displayed on a plurality of P.
pastoris cells as described above in Example 1. The fibronectin
library is modified to contain an MFalpha/HSA hybrid leader
sequence fused to the N-terminus and a NorpA ligand sequence fused
to the C-terminus. The modified fibronectin library is then cloned
into the pPlCHOLl-1 vector. As in the examples above, the
expression of the fibronectin library is under the control of the
AOX1 promoter. P. pastoris cells are transformed with the
pPlCHOLl-1 vectors expressing the fibronectin library and the
vectors expressing Aga1p and Aga2p-InaD. Expression of the
components is induced by the addition of methanol to the cells, and
the fibronectin library is displayed on a plurality of P. pastoris
cells.
[0107] Screening of Display Library
[0108] The yeast display fibronectin library is screened for
binding to a target protein of interest using one of marry methods
known in the an For example, the target protein is contacted with
the yeast display fibronectin library under conditions that allow
for the specific binding of the target protein to any members of
the library. All bound target protein is no immobilized on the
surface of a yeast cell. All unbound target protein is washed off.
The bound target protein is fluorescently labeled by methods well
known in the art, such as fluorescently labeled antibodies specific
for the target protein. The labeled target protein, now immobilized
on the surface of a yeast cell, is then detected using flow
cytometry, i.e. FACS. Yeast cells tint bind the labeled target
protein will fluoresce and are sorted from those yeast cells that
do not bind the target protein. The sorted yeast cells that have
bound the target protein are clonally expanded , and the clone line
or line containing members of the fibronectin library that bind the
target protein are determined.
[0109] Example 6
Screening of a Protein Library
[0110] Protein Library Display
[0111] A protein library is generated by methods well known in the
art, such as use of error prone PCR, use of random priming
techniques, or use of computational techniques. The protein library
is designed with appropriate restriction enzyme cleavage sites in
order to clone the library into yeast expression vectors.
[0112] The cloned protein library is displayed on a plurality of P.
pastoris cells as described above in Example 1. The protein library
is modified to contain an MFalpha/HSA hybrid leader sequence fused
to the N-terminus and a NorpA ligand sequence fused to the
C-terminus. The modified protein library is then cloned into the
pPlCHOLl-1 vector. As in the examples above, the expression of the
protein library is tinder the control of the AOX1 promoter. P.
pastoris cells are transformed with the pPlCHOLl-1 vectors
expressing the protein library and the vectors expressing Aga1p and
Aga2p-InaD. Expression of the components is induced by the addition
of methanol to the cells, and the protein library is displayed On a
plurality of P. pastoris cells.
[0113] Screening of Display Library
[0114] The yeast display protein library is screened for binding,
to a target protein of interest using one of many methods known in
the art. For example, the target protein is contacted with the
yeast display protein library under conditions that allow for the
specific binding of the target protein to any members of the
library. All bound target protein is now immobilized on the surface
of a yeast cell. All unbound target protein is washed off. The
bound target protein is fluorescently labeled by methods well known
in the art, such as fluorescently labeled antibodies specific for
the target protein. The labeled target protein, now immobilized on
the surface of a yeast cell, is then detected using flow cytometry,
i.e. FACS. Yeast cells that bind the labeled target protein will
fluoresce and are sorted from those yeast cells that do not hind
the target protein. The sorted yeast cells that have bound the
target protein are clonally expanded, and the clone line or line
containing members of the protein library that bind the target
protein are determined.
Example 7
Screening of a Fibronectin or HSA Libraries
Fibronectin or HSA Library Display
[0115] A fibronectin or HSA library is generated by methods well
known in the art, such as use of error prone PCR, use of random
priming techniques, or use of computational techniques. The
libraries are designed With appropriate restriction enzyme cleavage
sites in order to clone the libraries into yeast expression vectors
(See FIG. 8 and SEQ ID NO: 10).
[0116] The cloned fibronectin library or HSA library is displayed
on a plurality of P. pastoris cells as described above in Example
1. The fibronectin library is modified to contain an MFalpha/HSA
hybrid leader sequence used to the N-terminus and a NorpA ligand
sequence fused to the C-terminus (pYS HSA_MFalpha1 Fn10 NorpA). The
HSA library is modified to contain a MFalpha CT (C-Terminal) It is
a left-over of the original Invitrogen vector used for the
constructions. If you look at the vector map (e.g. FIG. 13) you can
see a c-terminal v5 and 6xhis sequence of the c-terminus of the
insert. I've placed a stop in front of it and it is not translated
in the final displayed protein leader sequence (pYS6/CT HSA-NorpA).
The modified fibronectin or HSA library is then cloned into the pYS
vector. The expression of the fibronectin or HSA library is under
the control of the T7 promoter. P. pastoris cells are transformed
with the pYS vectors expressing the fibronectin or HSA library and
the vectors expressing Aga1p and Aga2p-InaD, Expression of the
components is induced by the addition of methanol to the cells.,
and, the fibronectin or HSA library is displayed on a plurality of
P. pastoris cells.
[0117] (a) FACS Analysis of Protein Surface Expression,
[0118] Yeast cells expressing either fibronectin (pYS HSA_MFalpha1
Fn10 NorpA) or HSA (pYS6/CT HSA-NorpA) sere stained with anti-myc
antibody, followed by APC labeled secondary anti-mouse antibody and
then subjected to FACS analysis. The results of the analysis are
shown in FIGS. 9A-E. Specifically, FIG. 9A is the control sample
showing unstained yeast cells; FIG. 9B is a sample of uninduced
yeast cells expressing fibronectin; FIG. 9C is a sample of induced
yeast cells expressing fibronectin, showing a shift of cells
compared with the uninduced cells; FIG. 9D is a sample of uninduced
yeast cells expressing HSA: and FIG. 9E is a sample of induced
yeast cells expressing HSA, again showing a shill of cells compared
with the uninduced cells. These results clearly demonstrate that
the yeast display system is able to express fibronectin molecules,
and proteins, such as HSA.
[0119] b) Fluorometric Mircovolume Assay Technology (FMAT, Perkin
Elmer)Analysis of Yeast Expressing Fibronectin or HSA
[0120] Yeast cells expressing either fibronectin (plasmid) or HSA
(plasmid) were also analyzed by FMAT by staining with anti-myc
antibody and APC labeled secondary anti-mouse antibody. The samples
were then subjected to FMAT confocal fluorescence microscopy and
shown in FIGS. 10A-E. Those colonies that express fibronectin or
HSA appear as white dots against a black background. Specifically,
FIG. 10A is the control sample showing unstained yeast cells and
appears entirely black; FIG. 10B is a sample of uninduced yeast
cells expressing fibronectin. The uninduced yeast cells do not
produce fibronectin and are not detected (image appears black):
FIG. 10C is a sample of induced yeast cells expressing fibronectin.
In this instance, induction leads to the fibronectin with a myc tag
being expressed and detected using the anti-myc antibody.
Subsequent detection with the APC secondary anti-mouse antibody and
FMAT confocal fluorescence microscopy results in visible white
colonies being detected. FIG. 10D is a sample of uninduced yeast
cells expressing HSA, as before the uninduced yeast cells do not
produce fibronectin and the image appears black; and FIG. 10E is a
sample of induced yeast cells expressing HSA, again showing small
white colonies compared with the uninduced cells. These results
further confirm that the yeast display system is able to express
fibronectin molecule and proteins, such as HSA.
Example 8
Screening of Single Chain Fv Libraries
Single Chain Fv Library Display
[0121] A single chain scFv library is generated by methods well
known in the art, such as use of error prone PCR, use of random
priming techniques, or use of computational techniques. The
libraries are designed with appropriate restriction enzyme cleavage
sites in order to clone the libraries into yeast expression vectors
(See FIG. II and SEQ ID NO 11).
[0122] The cloned scFv lysozyme library is displayed on a plurality
of P. pastoris cells as described above in Example 1. The scFv
library is modified to contain an MFalpha leader sequence fused to
the N-terminus and a NorpA ligand sequence fused to the C-terminus
(pYS6/C.T* MFalpha1-scFv lysozyme-NorpA). The modified scFv
lysozyme library is then cloned into the pYS vector. The expression
of the scFv lysozyme is under the control of the 17 promoter. P.
pastoris cells are transformed with the pYS vectors expressing the
scFv lysozyme library and the vectors expressing Aga1p and
Aga2p-InaD. Expression of the components is induced by the addition
of methanol to the cells, and the fibronectin or HSA library is
displayed on a plurality of P. pastoris cells.
[0123] (a) FACS Analysis of Protein Surface Expression.
[0124] Yeast cells expressing the scFv lysozyme were stained with
anti-myc antibody, followed by APC labeled secondary anti-mouse
antibody and then subjected to FACS analysis.
[0125] (b) FMAT Analysis of Yeast Expressing Fibronectin or HSA
[0126] Yeast cells expressing the scFv lysozyme were also analyzed
by FMAT by staining with anti-myc antibody and APC labeled
secondary anti-mouse antibody. The samples were then subjected to
FMAT confocal fluorescence microscopy.
Example 9
Screening of Protein Libraries with a Reverse System
Protein Library Display
[0127] In the Example, the yeast display system described herein is
reversed such that NorpA is fused to Aga2 and the InaD is fused to
Aga1. A protein library is generated by methods well known in the
art, such as use of error prone PCR, use of random priming
techniques, or use of computational techniques. The libraries are
designed with appropriate restriction enzyme cleavage sites in
order to clone the libraries into yeast expression vectors (See
FIGS. 12 and 13 and SEQ ID NOs: 12 and 13).
[0128] The protein display system for P. pastoris was developed to
display a fibronectin type III domain (Fn10), by fusing a leader
sequence (MFalpha1)at N-terminus of InaD and fusing the Fn10 to its
C-terminus. One expressed, the Fn10 was secreted from the cell and
the PDZ domain of InaD bound to the NorpA ligand through disulfide
bonds. The NorpA as fused to the C-terminus of the Agap2 protein.
The Aga2p-NorpA fusion protein served as the adapter protein, and
the N-.sup.-terminal Aga2p bound Aga1p, which was immobilized on
the surface of the cell. Aga2p bound specifically to Aga1p through
disulfide bonds.
[0129] The three component system consisting of the Aga2p-NorpA
fusion protein, the Aga1-InaD fusion protein, and the Aga1p cell
surface protein were cloned into pPD and pYS expression vectors
respectively, under the control of a Gal inducible promoter.
[0130] The inducible promoter used was the Gal1 promoter, which is
induced by galactose. Thus when galactose was added to yeast cells
transformed with the vectors, the three proteins were expressed.
Aga1p as expressed on the surface of the cell. Aga2p-NorpA as
localized to the cell surface where the N-terminal region of
Aga2p-NorpA bound to Aga1p. Fn10-InaD was localized to the
secretory pathway, Was secreted front the cell, and bound NorpA via
the C-terminal InaD ligand.
TABLE-US-00001 pPIC3.5 AGA1 (956 bp-31336 bp, direct) 242aa (SEQ ID
NO 2) MTLSFAHFTY LFTILLGLTN IALASDPETI LVTITKTNDA NGVVTTTVSP
ALVSTSTIVQ AGTTTLYTTW CPLTVSTSSA AEISPSISYA TTLSRFSTLT LSTEVCSHEA
CPSSSTLPTT TLSVTSKFTS YICPTCHTTA ISSLSEVGTT TVVSSSAIEP SSASIISPVT
STLSSTTSSN PTTTSLSSTS TSPSSTSTSP SSTSTSSSST STSSSSTSTS SSSTSTSPSS
TSTSSSLTST SSSSTSTSQS STSTSSSSTS TSPSSTSTSS SSTSTSPSSK STSASSTSTS
SYSTSTSPSL TSSSPTLAST SPSSTSISST FTDSTSSLGS SIASSSTSVS LYSPSTPVYS
VPSTSSNVAT PSMTSSTVET TVSSQSSSEY ITKSSISTTI PSFSMSTYFT TVSGVTTMYT
TWCPYSSESE TSTLTSMHET VTTDATVCTH ESCMPSQTTS LITSSIKMST KNVATSVSTS
TVESSYACST CAETSHSYSS VQTASSSSVT QQTTSTKSWV SSMTTSDEDF NKHATGKYHV
TSSGTSTIST SVSEATSTSS IDSESQEQSS HLLSTSVLSS SSLSATLSSD STILLFSSVS
SLSVEQSPVT TLQISSTSEI LQPTSSTAIA TISASTSSLS ATSISTPSTS VESTIESSSL
TPTVSSIFLS SSSAPSSLQT SVTTTEVSTT SISIQYQTSS MVTISQYMGS GSQTRLPLGK
LVFAIMAVAC NVIFS pPIC6 A AGA2-InaD (941 bp-1648 bp, direct) 78aa
(SEQ ID NO: 3) MQLLRCFSIF SVIASVLAQE LTTICEQIPS PTLESTPYSL
STTTILANGK AMQGVFEYYK SVTFVSNCGS HPSTTSKGSP INTQYVFKLL QASGGGGSGG
GGSGGGGSAS MTGGQQMGRE NLYFQGVPGS SVVSRAGELI HMVTLDKTGK KSFGICIVRG
EVKDSPNTKT TGIFIKGIVP DSPAHLCGRL KVGDRILSLN GKDVRNSTEQ AVIDLIKEAD
FKIELEIQTF DK pPICHOLI-1_MFalpha1Hsa-Fn10-NorpA (884 bp-1441 bp,
direct) 62aa (SEQ ID NO 4) MKWVSFISLL FLFSSAYSRS LDKRENLYFQ
GGSVSDVPRD LEVVAATPTS LLISWDAPAV TVRYYRITYG ETGGNSPVQE FTVPGSKSTA
TISGLKPGVD YTITVYAVTG RGDSPASSKP ISINYRTEFE NLYFQGSGGG GEQKLISEED
LHHHHHHPST PPTPSPSTPP TPSPSYKTQG KTEFCA pPICHOL1-C
Mfalpha1Hsa-Fn10-NorpA (691 bp-1248 bp, direct) 62aa (SEQ ID NO: 5)
MXWVSFISLL FLFSSAYSRS LDKRENLYFQ GGSVSDVPRD LEVVAATPTS LLISWDAPAV
TVRYYRITYG ETGGNSPVQE FTVPGSKSTA TISGLKPGVD YTITVYAVTG RGDSPASSKP
ISINYRTEFE NLYFQGSGGG GEQKLISEED LHHHHHHPST PPTPSPSTPP TPSPSYKTQG
KTEFCA pYD_NBC1_Aga2-InaD (534 bp-1235 bp, direct) 78aa (SEQ. ID
NO: 6) MQLLRCFSIF SVIASVLAQE LTTICEQIPS PTLESTPYSL STTTILANGK
AMQGVFEYYK SVTFVSNCGS HPSTTSKGSP INTQYVFKLL QASGGGGSGG GGSGGGGSAS
MTGGQQMGRE NLYFQGVPGS SVVSRAGELI HMVTLDKTGK KSFGICIVRG EVKDSPNTKT
TGIFIKGIVP DSPAHLCGRL KVGDRILSLN GKDVRNSTEQ AVIDLIKEAD FKIELEIQTF
DK pYS6/CT_HSA _MFalpha1_Fn10_NorpA (513 bp-1080 bp, direct) 63aa
(SEQ ID NO: 7) MKWVSFISLL FLFSSAYSRS LDKRENLYFQ GGSVSDVPRD
LEVVAATPTS LLISWDAPAV TVRYYRITYG ETGGNSPVQE FTVPGSKSTA TISGLKPGVD
YTITVYAVTG RGDSPASSKP ISINYRTEFE NLYFQGSGGG GEQKLISEED LHHHHHHPST
PPTPSPSTPP TPSPSYNTQG KTEFCA InaD PDZ domain amino acid sequence
(InaD as 11-107) (SEQ ID NO 8) AGELIHMVTL DKTGKKSFGI CIVRGEVKDS
PNTKTTGIFI KGIVPDSPAH LCGRLKVGDR ILSLNGKDVR NSTEQAVIDL IKEADFKIEL
EIQTFDK NorpA C-terminal 11 amino acids including EFCA motif (SEQ
ID NO: 9) YKTQGKTEFC A pYS6/CT* MFalpha HSA-NorpA (SEQ ID NO: 10)
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST
NNGLLFINTTIASIAAKEEGVSLEKREAEAASDAHKSEVAERFKDLGEENFKALVLIAF
AQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGE
MADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARR
HPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ
KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKY
ICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEA
KDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLV
EEPQNLIKQNCELFEQLGEYKPQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKH
PEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP
KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
CKADDKETCFAEEGKKLVAASQAALGLGSENLYFQGSGGGGEQKLISEEDLHHHHHHHH
PSTPPTPSPSTPPTPSPSYKTQGKTEFCA. pYS6/CT* MFalpha1-scFv
lysozyme-NorpA (SEQ ID NO 11)
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNS
TNNGLLFINTTIASIAAKEEGVSLEKREAEAASQVKLQQSGAELVKPGASVKLSCTASG
FNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSS
LTSEDTAVYYCARWDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSMYT
SLGERVTITCKASQDINSYLRWFQQKPGKSPKTLIYYATSLADGVPSRFSGSGSGQDYS
LTISSLESDDTTTYYCLQHGESPYTEGGGTKLEIKRAAAEQKLISEEDLNGSENLYFQG
SGGGGEQKLISEEDLHHHHHHHHPSTPPTPSPSTPPTPSPSYKTQGKTEFCA
pYD_NBC1_Aga2-NorpA (SEQ ID NO: 12)
MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYY
KSVTFVSNCGSHPSTTSKGSPINTQYVFKLLQASGGGGSGGGGSYKTQGKTEFCA pYS6/Cf*
MFalpha1-InaD-Fn10 (507 bp-1487 bp, direct) 109aa (SEQ ID NO: 13)
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNST
NNGLLFINTTIASIAAKEEGVSLEKREAEAASAGELIHMVTLDKTGKKSFGICIVRGEV
KDSPNTKTTGIFIKGIVPDSPAHLCGRLKVGDRILSLNGKDVRNSTEQAVIDLIKEADF
KIELEIQTFDKSGGGGEQKLISEEDLHHHHHHPSTPPTPSPSTPPTPSPENLYFQGVSD
VPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGL
KPGVDYTITVYAVTGRGDSPASSKPISINYRT
Sequence CWU 1
1
13124PRTArtificial SequenceSaccharomyces cerevisiae 1Met Leu Leu
Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys1 5 10 15Ile Ser
Ala Asp Ala His Lys Ser 202725PRTArtificial SequencepPIC3.5 AGA1
2Met Thr Leu Ser Phe Ala His Phe Thr Tyr Leu Phe Thr Ile Leu Leu1 5
10 15Gly Leu Thr Asn Ile Ala Leu Ala Ser Asp Pro Glu Thr Ile Leu
Val 20 25 30Thr Ile Thr Lys Thr Asn Asp Ala Asn Gly Val Val Thr Thr
Thr Val 35 40 45Ser Pro Ala Leu Val Ser Thr Ser Thr Ile Val Gln Ala
Gly Thr Thr 50 55 60Thr Leu Tyr Thr Thr Trp Cys Pro Leu Thr Val Ser
Thr Ser Ser Ala65 70 75 80Ala Glu Ile Ser Pro Ser Ile Ser Tyr Ala
Thr Thr Leu Ser Arg Phe 85 90 95Ser Thr Leu Thr Leu Ser Thr Glu Val
Cys Ser His Glu Ala Cys Pro 100 105 110Ser Ser Ser Thr Leu Pro Thr
Thr Thr Leu Ser Val Thr Ser Lys Phe 115 120 125Thr Ser Tyr Ile Cys
Pro Thr Cys His Thr Thr Ala Ile Ser Ser Leu 130 135 140Ser Glu Val
Gly Thr Thr Thr Val Val Ser Ser Ser Ala Ile Glu Pro145 150 155
160Ser Ser Ala Ser Ile Ile Ser Pro Val Thr Ser Thr Leu Ser Ser Thr
165 170 175Thr Ser Ser Asn Pro Thr Thr Thr Ser Leu Ser Ser Thr Ser
Thr Ser 180 185 190Pro Ser Ser Thr Ser Thr Ser Pro Ser Ser Thr Ser
Thr Ser Ser Ser 195 200 205Ser Thr Ser Thr Ser Ser Ser Ser Thr Ser
Thr Ser Ser Ser Ser Thr 210 215 220Ser Thr Ser Pro Ser Ser Thr Ser
Thr Ser Ser Ser Leu Thr Ser Thr225 230 235 240Ser Ser Ser Ser Thr
Ser Thr Ser Gln Ser Ser Thr Ser Thr Ser Ser 245 250 255Ser Ser Thr
Ser Thr Ser Pro Ser Ser Thr Ser Thr Ser Ser Ser Ser 260 265 270Thr
Ser Thr Ser Pro Ser Ser Lys Ser Thr Ser Ala Ser Ser Thr Ser 275 280
285Thr Ser Ser Tyr Ser Thr Ser Thr Ser Pro Ser Leu Thr Ser Ser Ser
290 295 300Pro Thr Leu Ala Ser Thr Ser Pro Ser Ser Thr Ser Ile Ser
Ser Thr305 310 315 320Phe Thr Asp Ser Thr Ser Ser Leu Gly Ser Ser
Ile Ala Ser Ser Ser 325 330 335Thr Ser Val Ser Leu Tyr Ser Pro Ser
Thr Pro Val Tyr Ser Val Pro 340 345 350Ser Thr Ser Ser Asn Val Ala
Thr Pro Ser Met Thr Ser Ser Thr Val 355 360 365Glu Thr Thr Val Ser
Ser Gln Ser Ser Ser Glu Tyr Ile Thr Lys Ser 370 375 380Ser Ile Ser
Thr Thr Ile Pro Ser Phe Ser Met Ser Thr Tyr Phe Thr385 390 395
400Thr Val Ser Gly Val Thr Thr Met Tyr Thr Thr Trp Cys Pro Tyr Ser
405 410 415Ser Glu Ser Glu Thr Ser Thr Leu Thr Ser Met His Glu Thr
Val Thr 420 425 430Thr Asp Ala Thr Val Cys Thr His Glu Ser Cys Met
Pro Ser Gln Thr 435 440 445Thr Ser Leu Ile Thr Ser Ser Ile Lys Met
Ser Thr Lys Asn Val Ala 450 455 460Thr Ser Val Ser Thr Ser Thr Val
Glu Ser Ser Tyr Ala Cys Ser Thr465 470 475 480Cys Ala Glu Thr Ser
His Ser Tyr Ser Ser Val Gln Thr Ala Ser Ser 485 490 495Ser Ser Val
Thr Gln Gln Thr Thr Ser Thr Lys Ser Trp Val Ser Ser 500 505 510Met
Thr Thr Ser Asp Glu Asp Phe Asn Lys His Ala Thr Gly Lys Tyr 515 520
525His Val Thr Ser Ser Gly Thr Ser Thr Ile Ser Thr Ser Val Ser Glu
530 535 540Ala Thr Ser Thr Ser Ser Ile Asp Ser Glu Ser Gln Glu Gln
Ser Ser545 550 555 560His Leu Leu Ser Thr Ser Val Leu Ser Ser Ser
Ser Leu Ser Ala Thr 565 570 575Leu Ser Ser Asp Ser Thr Ile Leu Leu
Phe Ser Ser Val Ser Ser Leu 580 585 590Ser Val Glu Gln Ser Pro Val
Thr Thr Leu Gln Ile Ser Ser Thr Ser 595 600 605Glu Ile Leu Gln Pro
Thr Ser Ser Thr Ala Ile Ala Thr Ile Ser Ala 610 615 620Ser Thr Ser
Ser Leu Ser Ala Thr Ser Ile Ser Thr Pro Ser Thr Ser625 630 635
640Val Glu Ser Thr Ile Glu Ser Ser Ser Leu Thr Pro Thr Val Ser Ser
645 650 655Ile Phe Leu Ser Ser Ser Ser Ala Pro Ser Ser Leu Gln Thr
Ser Val 660 665 670Thr Thr Thr Glu Val Ser Thr Thr Ser Ile Ser Ile
Gln Tyr Gln Thr 675 680 685Ser Ser Met Val Thr Ile Ser Gln Tyr Met
Gly Ser Gly Ser Gln Thr 690 695 700Arg Leu Pro Leu Gly Lys Leu Val
Phe Ala Ile Met Ala Val Ala Cys705 710 715 720Asn Val Ile Phe Ser
7253232PRTArtificial SequencepPIC6 A AGA2-InaD 3Met Gln Leu Leu Arg
Cys Phe Ser Ile Phe Ser Val Ile Ala Ser Val1 5 10 15Leu Ala Gln Glu
Leu Thr Thr Ile Cys Glu Gln Ile Pro Ser Pro Thr 20 25 30Leu Glu Ser
Thr Pro Tyr Ser Leu Ser Thr Thr Thr Ile Leu Ala Asn 35 40 45Gly Lys
Ala Met Gln Gly Val Phe Glu Tyr Tyr Lys Ser Val Thr Phe 50 55 60Val
Ser Asn Cys Gly Ser His Pro Ser Thr Thr Ser Lys Gly Ser Pro65 70 75
80Ile Asn Thr Gln Tyr Val Phe Lys Leu Leu Gln Ala Ser Gly Gly Gly
85 90 95Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Met
Thr 100 105 110Gly Gly Gln Gln Met Gly Arg Glu Asn Leu Tyr Phe Gln
Gly Val Pro 115 120 125Gly Ser Ser Val Val Ser Arg Ala Gly Glu Leu
Ile His Met Val Thr 130 135 140Leu Asp Lys Thr Gly Lys Lys Ser Phe
Gly Ile Cys Ile Val Arg Gly145 150 155 160Glu Val Lys Asp Ser Pro
Asn Thr Lys Thr Thr Gly Ile Phe Ile Lys 165 170 175Gly Ile Val Pro
Asp Ser Pro Ala His Leu Cys Gly Arg Leu Lys Val 180 185 190Gly Asp
Arg Ile Leu Ser Leu Asn Gly Lys Asp Val Arg Asn Ser Thr 195 200
205Glu Gln Ala Val Ile Asp Leu Ile Lys Glu Ala Asp Phe Lys Ile Glu
210 215 220Leu Glu Ile Gln Thr Phe Asp Lys225 2304186PRTArtificial
SequencepPICHOLI-1_MFalpha1Hsa-Fn10-NorpA 4Met Lys Trp Val Ser Phe
Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Ser Leu
Asp Lys Arg Glu Asn Leu Tyr Phe Gln Gly Gly 20 25 30Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro 35 40 45Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr 50 55 60Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu65 70 75
80Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys
85 90 95Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg
Gly 100 105 110Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg Thr Glu 115 120 125Phe Glu Asn Leu Tyr Phe Gln Gly Ser Gly Gly
Gly Gly Glu Gln Lys 130 135 140Leu Ile Ser Glu Glu Asp Leu His His
His His His His Pro Ser Thr145 150 155 160Pro Pro Thr Pro Ser Pro
Ser Thr Pro Pro Thr Pro Ser Pro Ser Tyr 165 170 175Lys Thr Gln Gly
Lys Thr Glu Phe Cys Ala 180 1855186PRTArtificial SequencepPICHOLI-C
Mfalpha1Hsa-Fn10-NorpA 5Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Ser Leu Asp Lys Arg Glu Asn
Leu Tyr Phe Gln Gly Gly 20 25 30Ser Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro 35 40 45Thr Ser Leu Leu Ile Ser Trp Asp
Ala Pro Ala Val Thr Val Arg Tyr 50 55 60Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu65 70 75 80Phe Thr Val Pro Gly
Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys 85 90 95Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly 100 105 110Asp Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu 115 120
125Phe Glu Asn Leu Tyr Phe Gln Gly Ser Gly Gly Gly Gly Glu Gln Lys
130 135 140Leu Ile Ser Glu Glu Asp Leu His His His His His His Pro
Ser Thr145 150 155 160Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr
Pro Ser Pro Ser Tyr 165 170 175Lys Thr Gln Gly Lys Thr Glu Phe Cys
Ala 180 1856232PRTArtificial SequencepYD_NBC1_Aga2-InaD 6Met Gln
Leu Leu Arg Cys Phe Ser Ile Phe Ser Val Ile Ala Ser Val1 5 10 15Leu
Ala Gln Glu Leu Thr Thr Ile Cys Glu Gln Ile Pro Ser Pro Thr 20 25
30Leu Glu Ser Thr Pro Tyr Ser Leu Ser Thr Thr Thr Ile Leu Ala Asn
35 40 45Gly Lys Ala Met Gln Gly Val Phe Glu Tyr Tyr Lys Ser Val Thr
Phe 50 55 60Val Ser Asn Cys Gly Ser His Pro Ser Thr Thr Ser Lys Gly
Ser Pro65 70 75 80Ile Asn Thr Gln Tyr Val Phe Lys Leu Leu Gln Ala
Ser Gly Gly Gly 85 90 95Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Ala Ser Met Thr 100 105 110Gly Gly Gln Gln Met Gly Arg Glu Asn
Leu Tyr Phe Gln Gly Val Pro 115 120 125Gly Ser Ser Val Val Ser Arg
Ala Gly Glu Leu Ile His Met Val Thr 130 135 140Leu Asp Lys Thr Gly
Lys Lys Ser Phe Gly Ile Cys Ile Val Arg Gly145 150 155 160Glu Val
Lys Asp Ser Pro Asn Thr Lys Thr Thr Gly Ile Phe Ile Lys 165 170
175Gly Ile Val Pro Asp Ser Pro Ala His Leu Cys Gly Arg Leu Lys Val
180 185 190Gly Asp Arg Ile Leu Ser Leu Asn Gly Lys Asp Val Arg Asn
Ser Thr 195 200 205Glu Gln Ala Val Ile Asp Leu Ile Lys Glu Ala Asp
Phe Lys Ile Glu 210 215 220Leu Glu Ile Gln Thr Phe Asp Lys225
2307186PRTArtificial SequencepYS6/CT_HSA_MFalpha1_Fn10_NorpA 7Met
Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10
15Tyr Ser Arg Ser Leu Asp Lys Arg Glu Asn Leu Tyr Phe Gln Gly Gly
20 25 30Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro 35 40 45Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val
Arg Tyr 50 55 60Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu65 70 75 80Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr
Ile Ser Gly Leu Lys 85 90 95Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr
Ala Val Thr Gly Arg Gly 100 105 110Asp Ser Pro Ala Ser Ser Lys Pro
Ile Ser Ile Asn Tyr Arg Thr Glu 115 120 125Phe Glu Asn Leu Tyr Phe
Gln Gly Ser Gly Gly Gly Gly Glu Gln Lys 130 135 140Leu Ile Ser Glu
Glu Asp Leu His His His His His His Pro Ser Thr145 150 155 160Pro
Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Tyr 165 170
175Lys Thr Gln Gly Lys Thr Glu Phe Cys Ala 180 185897PRTUnknownInaD
PDZ domain amino acid sequence 8Ala Gly Glu Leu Ile His Met Val Thr
Leu Asp Lys Thr Gly Lys Lys1 5 10 15Ser Phe Gly Ile Cys Ile Val Arg
Gly Glu Val Lys Asp Ser Pro Asn 20 25 30Thr Lys Thr Thr Gly Ile Phe
Ile Lys Gly Ile Val Pro Asp Ser Pro 35 40 45Ala His Leu Cys Gly Arg
Leu Lys Val Gly Asp Arg Ile Leu Ser Leu 50 55 60Asn Gly Lys Asp Val
Arg Asn Ser Thr Glu Gln Ala Val Ile Asp Leu65 70 75 80Ile Lys Glu
Ala Asp Phe Lys Ile Glu Leu Glu Ile Gln Thr Phe Asp 85 90
95Lys911PRTUnknownNorpA C-terminal 9Tyr Lys Thr Gln Gly Lys Thr Glu
Phe Cys Ala1 5 1010737PRTArtificial SequencepYS6/CT* MFalpha
HSA-NorpA 10Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala
Ser Ser1 5 10 15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu
Thr Ala Gln 20 25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu
Glu Gly Asp Phe 35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr
Asn Asn Gly Leu Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala
Ala Lys Glu Glu Gly Val65 70 75 80Ser Leu Glu Lys Arg Glu Ala Glu
Ala Ala Ser Asp Ala His Lys Ser 85 90 95Glu Val Ala His Arg Phe Lys
Asp Leu Gly Glu Glu Asn Phe Lys Ala 100 105 110Leu Val Leu Ile Ala
Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu 115 120 125Asp His Val
Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 130 135 140Val
Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu145 150
155 160Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr
Gly 165 170 175Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg
Asn Glu Cys 180 185 190Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
Pro Arg Leu Val Arg 195 200 205Pro Glu Val Asp Val Met Cys Thr Ala
Phe His Asp Asn Glu Glu Thr 210 215 220Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg Arg His Pro Tyr Phe225 230 235 240Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe 245 250 255Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 260 265
270Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg
275 280 285Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe
Lys Ala 290 295 300Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys
Ala Glu Phe Ala305 310 315 320Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys Val His Thr Glu Cys 325 330 335Cys His Gly Asp Leu Leu Glu
Cys Ala Asp Asp Arg Ala Asp Leu Ala 340 345 350Lys Tyr Ile Cys Glu
Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu 355 360 365Cys Cys Glu
Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val 370 375 380Glu
Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe385 390
395 400Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp
Val 405 410 415Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His
Pro Asp Tyr 420 425 430Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
Tyr Glu Thr Thr Leu 435 440 445Glu Lys Cys Cys Ala Ala Ala Asp Pro
His Glu Cys Tyr Ala Lys Val 450 455 460Phe Asp Glu Phe Lys Pro Leu
Val Glu Glu Pro Gln Asn Leu Ile Lys465 470 475 480Gln Asn Cys Glu
Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn 485 490 495Ala Leu
Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro 500 505
510Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys
515 520 525Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys Ala Glu Asp Tyr Leu 530 535 540Ser Val Val Leu Asn Gln Leu
Cys Val Leu His Glu Lys Thr Pro Val545 550 555 560Ser Asp Arg Val
Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg 565 570 575Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu 580 585
590Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser
595 600 605Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu
Leu Val 610 615 620Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys
Ala Val Met Asp625 630 635 640Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys Ala Asp Asp Lys Glu 645 650 655Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val Ala Ala Ser Gln Ala 660 665 670Ala Leu Gly Leu Gly
Ser Glu Asn Leu Tyr Phe Gln Gly Ser Gly Gly 675 680 685Gly Gly Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu His His His His 690 695 700His
His His His Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro705 710
715 720Pro Thr Pro Ser Pro Ser Tyr Lys Thr Gln Gly Lys Thr Glu Phe
Cys 725 730 735Ala11405PRTArtificial SequencepYS6/CT* MFalpha1-scFv
lysozyme-NorpA 11Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe
Ala Ala Ser Ser1 5 10 15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu
Asp Glu Thr Ala Gln 20 25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser
Asp Leu Glu Gly Asp Phe 35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn
Ser Thr Asn Asn Gly Leu Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser
Ile Ala Ala Lys Glu Glu Gly Val65 70 75 80Ser Leu Glu Lys Arg Glu
Ala Glu Ala Ala Ser Gln Val Lys Leu Gln 85 90 95Gln Ser Gly Ala Glu
Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser 100 105 110Cys Thr Ala
Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met His Trp Val 115 120 125Lys
Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro 130 135
140Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly Lys Ala
Thr145 150 155 160Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu
Gln Leu Ser Ser 165 170 175Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg Trp Asp Trp 180 185 190Tyr Phe Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Gly 195 200 205Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 210 215 220Glu Leu Thr Gln
Ser Pro Ser Ser Met Tyr Thr Ser Leu Gly Glu Arg225 230 235 240Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Arg 245 250
255Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile Tyr Tyr
260 265 270Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly 275 280 285Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu
Glu Ser Asp Asp 290 295 300Thr Thr Thr Tyr Tyr Cys Leu Gln His Gly
Glu Ser Pro Tyr Thr Phe305 310 315 320Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Ala Ala Ala Glu Gln Lys 325 330 335Leu Ile Ser Glu Glu
Asp Leu Asn Gly Ser Glu Asn Leu Tyr Phe Gln 340 345 350Gly Ser Gly
Gly Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 355 360 365His
His His His His His His His Pro Ser Thr Pro Pro Thr Pro Ser 370 375
380Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Tyr Lys Thr Gln Gly
Lys385 390 395 400Thr Glu Phe Cys Ala 40512114PRTArtificial
SequencepYD_NBC1_Aga2-NorpA 12Met Gln Leu Leu Arg Cys Phe Ser Ile
Phe Ser Val Ile Ala Ser Val1 5 10 15Leu Ala Gln Glu Leu Thr Thr Ile
Cys Glu Gln Ile Pro Ser Pro Thr 20 25 30Leu Glu Ser Thr Pro Tyr Ser
Leu Ser Thr Thr Thr Ile Leu Ala Asn 35 40 45Gly Lys Ala Met Gln Gly
Val Phe Glu Tyr Tyr Lys Ser Val Thr Phe 50 55 60Val Ser Asn Cys Gly
Ser His Pro Ser Thr Thr Ser Lys Gly Ser Pro65 70 75 80Ile Asn Thr
Gln Tyr Val Phe Lys Leu Leu Gln Ala Ser Gly Gly Gly 85 90 95Gly Ser
Gly Gly Gly Gly Ser Tyr Lys Thr Gln Gly Lys Thr Glu Phe 100 105
110Cys Ala13327PRTArtificial SequencepYS6/CT* MFalpha1-InaD-Fn10
13Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1
5 10 15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala
Gln 20 25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly
Asp Phe 35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn
Gly Leu Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys
Glu Glu Gly Val65 70 75 80Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala
Ser Ala Gly Glu Leu Ile 85 90 95His Met Val Thr Leu Asp Lys Thr Gly
Lys Lys Ser Phe Gly Ile Cys 100 105 110Ile Val Arg Gly Glu Val Lys
Asp Ser Pro Asn Thr Lys Thr Thr Gly 115 120 125Ile Phe Ile Lys Gly
Ile Val Pro Asp Ser Pro Ala His Leu Cys Gly 130 135 140Arg Leu Lys
Val Gly Asp Arg Ile Leu Ser Leu Asn Gly Lys Asp Val145 150 155
160Arg Asn Ser Thr Glu Gln Ala Val Ile Asp Leu Ile Lys Glu Ala Asp
165 170 175Phe Lys Ile Glu Leu Glu Ile Gln Thr Phe Asp Lys Ser Gly
Gly Gly 180 185 190Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu His
His His His His 195 200 205His Pro Ser Thr Pro Pro Thr Pro Ser Pro
Ser Thr Pro Pro Thr Pro 210 215 220Ser Pro Glu Asn Leu Tyr Phe Gln
Gly Val Ser Asp Val Pro Arg Asp225 230 235 240Leu Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp 245 250 255Ala Pro Ala
Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 260 265 270Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser 275 280
285Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
290 295 300Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser
Lys Pro305 310 315 320Ile Ser Ile Asn Tyr Arg Thr 325
* * * * *