U.S. patent application number 15/564619 was filed with the patent office on 2018-09-13 for biosynthetic amyloid-based materials displaying functional protein sequences.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Anna Duraj-Thatte, Neel S. Joshi, Peter Q. Nguyen, Pichet Praveschotinunt.
Application Number | 20180258435 15/564619 |
Document ID | / |
Family ID | 57072695 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180258435 |
Kind Code |
A1 |
Joshi; Neel S. ; et
al. |
September 13, 2018 |
BIOSYNTHETIC AMYLOID-BASED MATERIALS DISPLAYING FUNCTIONAL PROTEIN
SEQUENCES
Abstract
Embodiments of the present disclosure are directed to methods of
genetically modifying bacteria to create amyloid-based materials,
such as biofilms created by amyloid fibers, having nonnative
functional polypeptides expressed thereon and connected thereto by
a linker domain optimized for functioning of the non-native
functional polypeptides. According to one aspect, the linker domain
is optimized for functioning of a CsgA protein as is it assembled
into an amyloid state and for functioning of the functional
polypeptide. Exemplary biofilms may include living bacterial cells,
non-living bacterial cells or combinations of living bacterial
cells and non-living bacterial cells. Methods of making biofilms
having non-native functional polypeptides attached thereto are
provided.
Inventors: |
Joshi; Neel S.; (Somerville,
MA) ; Nguyen; Peter Q.; (Malden, MA) ;
Duraj-Thatte; Anna; (Arlington, MA) ;
Praveschotinunt; Pichet; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
President and Fellows of Harvard College
Cambridge
MA
|
Family ID: |
57072695 |
Appl. No.: |
15/564619 |
Filed: |
April 6, 2016 |
PCT Filed: |
April 6, 2016 |
PCT NO: |
PCT/US2016/026161 |
371 Date: |
October 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62143560 |
Apr 6, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/62 20130101;
C12N 2800/101 20130101; C07K 14/245 20130101; C07K 2319/00
20130101; C07K 14/33 20130101; A61K 35/74 20130101; C12N 15/70
20130101; A61P 1/00 20180101; C07K 2317/24 20130101 |
International
Class: |
C12N 15/62 20060101
C12N015/62; C12N 15/70 20060101 C12N015/70; C07K 14/245 20060101
C07K014/245; A61P 1/00 20060101 A61P001/00; C07K 14/33 20060101
C07K014/33 |
Goverment Interests
STATEMENT OF GOVERNMENT INTERESTS
[0002] This invention was made with government support under
DMR-1410751 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A method of making a biofilm comprising proliferating a bacteria
cell including a nucleic acid sequence encoding a fusion of a CsgA
protein linked to a non-native functional polypeptide by a linker
to produce a population of bacteria cells expressing the fusion and
forming a biofilm having the non-native functional polypeptide
attached thereto, wherein the linker is a polypeptide comprising 7
or more amino acids attached to either the C terminus or the N
terminus of the CsgA protein.
2. The method of claim 1 wherein the nucleic acid sequence is
introduced into the bacteria cell.
3. The method of claim 1 wherein the bacteria cell is E. coli.
4. The method of claim 1 wherein the bacteria cell is a
non-pathogenic bacteria.
5. The method of claim 1 wherein the bacteria cell is Nissle strain
1917 (EcN), MG1655, LSR10 or PHL628.
6. The method of claim 1 wherein the bacteria cell includes a
genomic deletion of the CsgA gene.
7. The method of claim 1 wherein the bacteria cell has been
genetically modified to remove a nucleic acid or nucleic acids
encoding the CsgA protein.
8. The method of claim 1 wherein the bacteria cell has been
genetically modified to remove the CsgA protein.
9. The method of claim 1 wherein the bacteria cell lacks expression
of the natural CsgA gene is Nissle strain 1917 (EcN), MG1655, LSR10
or PHL628.
10. The method of claim 9 wherein the bacteria cell that lacks
expression of the natural CsgA gene is Nissle strain 1917 (EcN),
MG1655, LSR10 or PHL628.
11. The method of claim 1 wherein the bacteria cell is a knockout
for the CsgA gene.
12. The method of claim 1 wherein the linker is between 7 and 250
amino acids.
13. The method of claim 1 wherein the linker is a flexible linker
or a rigid linker.
14. The method of claim 1 wherein the linker is hydrophilic or
hydrophobic.
15. The method of claim 1 wherein the linker comprises a repeating
amino acid subunit.
16. The method of claim 1 wherein the linker comprises two
repeating amino acid subunits or more.
17. The method of claim 1 wherein the linker comprises 3 repeating
amino acid subunits or more.
18. The method of claim 1 wherein the linker comprises [GGGS].sub.n
wherein n is an integer from 2 to 20 (SEQ ID NO:12).
19. The method of claim 1 wherein the linker comprises [GGGS].sub.n
wherein n is an integer being 3, 6, or 12 (SEQ ID NO:5-7).
20. The method of claim 1 wherein the linker comprises [P].sub.n
wherein n is an integer from 1 to 30 (SEQ ID NO:13).
21. The method of claim 1 wherein the linker comprises [P].sub.n
wherein n is an integer being 12 or 24 (SEQ ID NO:8-9).
22. The method of claim 1 wherein the linker comprises
[EAAAK].sub.n wherein n is an integer from 1 to 15 (SEQ ID
NO:15).
23. The method of claim 1 wherein the linker comprises
[EAAAK].sub.n wherein n is an integer being 3 or 9 (SEQ ID
NO:10-11).
24. The method of claim 1 wherein the linker comprises one or more
of glycine, serine, alanine or leucine.
25. The method of claim 1 wherein the linker comprises one or more
of [GGGS].sub.n wherein n is an integer from 2 to 20 (SEQ ID
NO:12), [P].sub.n wherein n is an integer from 1 to 30 (SEQ ID
NO:13) or [EAAAK].sub.n wherein n is an integer from 1 to 15 (SEQ
ID NO:15).
26. The method of claim 1 wherein the linker is cleavable.
27. The method of claim 1 wherein the linker is cleavable by an
enzyme.
28. The method of claim 1 wherein the non-native functional
polypeptide is releasable.
29. The method of claim 1 wherein the linker is GGGSGGGSGGGS,
GGGSGGGSGGGSGGGSGGGSGGGS,
GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 5-7),
PPPPPPPPPPPP, PPPPPPPPPPPPPPPPPPPPPPPP (SEQ ID NO:8-9),
EAAAKEAAAKEAAAK, or EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK
(SEQ ID NO:10-11).
30. The method of claim 1 wherein the non-native functional
polypeptide is a therapeutic polypeptide, a diagnostic polypeptide,
a tissue-binding polypeptide, a cell-binding polypeptide, an
antimicrobial polypeptide, an anticancer polypeptide, an
anti-inflammatory polypeptide, a polymer binding polypeptide, a
metabolite binding polypeptide, a targeting polypeptide or a
polypeptide that is a first pair of a binding pair of
molecules.
31. The method of claim 1 wherein the bacteria cell is Nissle
strain 1917 (EcN) and the linker is [GGGS].sub.n wherein n is and
integer being 3, 6, or 12 (SEQ ID NO:5-7).
32. A non-naturally occurring fusion of a CsgA protein linked to a
non-native functional polypeptide by a linker, wherein the linker
comprises [GGGS], wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
33. A nucleic acid sequence encoding a fusion of a CsgA protein
linked to a non-native functional polypeptide by a linker, wherein
the linker comprises [GGGS].sub.n wherein n is an integer from 2 to
20 (SEQ ID NO:12).
34. A vector comprising a nucleic acid sequence encoding a fusion
of a CsgA protein linked to a non-native functional polypeptide by
a linker, wherein the linker comprises [GGGS].sub.n wherein n is an
integer from 2 to 20 (SEQ ID NO:12).
35. A bacteria cell including a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to a non-native
functional polypeptide by a linker, wherein the linker comprises
[GGGS].sub.n wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
36. A bacteria cell including a vector comprising a nucleic acid
sequence encoding a fusion of a CsgA protein linked to a non-native
functional polypeptide by a linker, wherein the linker comprises
[GGGS].sub.n wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
37. A bacteria cell expressing a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to a non-native
functional polypeptide by a linker, wherein the linker comprises
[GGGS].sub.n wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
38. A biofilm including a bacteria cell expressing a foreign
nucleic acid sequence encoding a fusion of a CsgA protein linked to
a non-native functional polypeptide by a linker, wherein the linker
comprises [GGGS].sub.n wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
39. A method of delivering a bacteria cell to a tissue within an
organism comprising introducing into the organism a bacteria cell
expressing a foreign nucleic acid sequence encoding a fusion of a
CsgA protein linked to a tissue binding polypeptide by a linker,
wherein the linker comprises [GGGS].sub.n wherein n is an integer
from 2 to 20 (SEQ ID NO:12); wherein the bacteria cell attaches to
the tissue by the tissue binding polypeptide thereby localizing the
bacteria cell to the tissue.
40. The method of claim 39 wherein the bacteria cell proliferates
and a biofilm including a population of bacteria cells is formed
that is attached to the tissue.
41. The method of claim 39 wherein the bacteria cell is Nissle
strain 1917 (EcN).
42. The method of claim 39 wherein the tissue binding polypeptide
is CP15, P8, A1, T18, TTF1, TTF2 or TTF3.
43. The method of claim 39 wherein the tissue within the organism
is gastrointestinal tract epithelial tissue.
44. The method of claim 39 wherein the tissue within the organism
is Peyer's Patches.
45. The method of claim 39 wherein the tissue within the organism
is an infection site.
46. The method of claim 39 wherein the tissue within the organism
is an injured area of epithelium.
47. The method of claim 39 wherein the tissue within the organism
is tumor tissue.
48. The method of claim 39 wherein the tissue within the organism
is a site of inflammation.
49. The method of claim 39 wherein the tissue within the organism
includes colon carcinoma cells.
50. The method of claim 39 wherein the tissue within the organism
is gut mucosa.
51. The method of claim 39 wherein the tissue within the organism
includes M cells.
52. A method of treating an organism with inflammation of the
gastrointestinal tract comprising introducing into the organism a
Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
tissue binding polypeptide by a linker, wherein the linker
comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID
NO:12); wherein the Nissle strain 1917 (EcN) bacteria cell attaches
to tissue of the gastrointestinal tract by the tissue binding
polypeptide thereby localizing the Nissle strain 1917 (EcN)
bacteria cell to the tissue in a manner to reduce the
inflammation.
53. The method of claim 52 wherein the Nissle strain 1917 (EcN)
bacteria cell proliferates and a biofilm including a population of
Nissle strain 1917 (EcN) bacteria cells is formed that is attached
to the tissue.
54. The method of claim 52 wherein the tissue binding polypeptide
is CP15, P8, A1, T18, TTF1, TTF2 or TTF3.
55. The method of claim 52 wherein the inflammation results from
inflammatory bowel disease.
56. The method of claim 52 wherein the inflammation results from
Crohn's disease or ulcerative colitis.
57. A method of treating an organism with inflammation of the
gastrointestinal tract comprising introducing into the organism a
Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to an
anti-inflammatory polypeptide by a linker, wherein the linker
comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID
NO:12); wherein the Nissle strain 1917 (EcN) bacteria cell
proliferates within the gastrointestinal tract and a biofilm
including a population of Nissle strain 1917 (EcN) bacteria cells
is formed that includes the anti-inflammatory polypeptide in a
manner to reduce the inflammation.
58. The method of claim 57 wherein the anti-inflammatory
polypeptide is TTF1, TTF2 or TTF3.
59. The method of claim 57 wherein the inflammation results from
inflammatory bowel disease or Crohn's disease.
60. The method of claim 57 wherein the anti-inflammatory
polypeptide is released.
61. The method of claim 57 wherein the linker is a cleavable linker
and the anti-inflammatory polypeptide is released by cleaving the
linker.
62. The method of claim 57 wherein the linker is a cleavable linker
and the anti-inflammatory polypeptide is released by cleaving the
linker with an enzyme.
63. The method of claim 57 wherein the linker is a cleavable linker
and the anti-inflammatory polypeptide is released by cleaving the
linker with a protease produced as a result of the
inflammation.
64. The method of claim 57 wherein the linker is a cleavable linker
and the anti-inflammatory polypeptide is released by cleaving the
linker with an MMP protease.
65. A method of treating an organism with cancer of the
gastrointestinal tract comprising introducing into the organism a
Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to an
anti-cancer polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);
wherein the Nissle strain 1917 (EcN) bacteria cell proliferates
within the gastrointestinal tract and a biofilm including a
population of Nissle strain 1917 (EcN) bacteria cells is formed
that includes the anti-cancer polypeptide in a manner to reduce
proliferation of the cancer.
66. The method of claim 65 wherein the anti-cancer polypeptide is a
growth inhibiting biologic.
67. The method of claim 65 wherein the anti-cancer polypeptide is
released.
68. The method of claim 65 wherein the linker is a cleavable linker
and the anti-cancer polypeptide is released by cleaving the
linker.
69. The method of claim 65 wherein the linker is a cleavable linker
and the anti-cancer polypeptide is released by cleaving the linker
with an enzyme.
70. The method of claim 65 wherein the linker is a cleavable linker
and the anti-cancer polypeptide is released by cleaving the linker
with an enzyme within the gastrointestinal tract.
71. A method of treating an organism with microbial pathogens in
the gastrointestinal tract comprising introducing into the organism
a Nissle strain 1917 (EcN) bacteria cell expressing a foreign
nucleic acid sequence encoding a fusion of a CsgA protein linked to
an anti-microbial polypeptide by a linker, wherein the linker
comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID
NO:12); wherein the Nissle strain 1917 (EcN) bacteria cell
proliferates within the gastrointestinal tract and a biofilm
including a population of Nissle strain 1917 (EcN) bacteria cells
is formed that includes the anti-microbial polypeptide in a manner
to reduce proliferation of the microbial pathogens.
72. The method of claim 71 wherein the microbial pathogen is
Clostridium difficile.
73. The method of claim 71 wherein the anti-microbial polypeptide
is coprisin, thurcin CD, a lanibiotic, nisin, actagardine, a
cathelicidin or LL-37.
74. The method of claim 71 wherein the anti-microbial polypeptide
is released.
75. The method of claim 71 wherein the linker is a cleavable linker
and the anti-microbial polypeptide is released by cleaving the
linker.
76. The method of claim 71 wherein the linker is a cleavable linker
and the anti-microbial polypeptide is released by cleaving the
linker with an enzyme.
77. The method of claim 71 wherein the linker is a cleavable linker
and the anti-microbial polypeptide is released by cleaving the
linker with an enzyme within the gastrointestinal tract.
78. The method of claim 71 wherein the linker is a cleavable linker
and the anti-microbial polypeptide is released by cleaving the
linker with protease CD2830 within the gastrointestinal tract.
79. The method of claim 71 wherein the linker is a cleavable linker
and the anti-microbial polypeptide is released when exposed to
microbial pathogen.
80. A method of delivering a diagnostic polypeptide to the
gastrointestinal tract of an organism comprising introducing into
the organism a Nissle strain 1917 (EcN) bacteria cell expressing a
foreign nucleic acid sequence encoding a fusion of a CsgA protein
linked to a diagnostic polypeptide by a linker, wherein the linker
comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID
NO:12); wherein the Nissle strain 1917 (EcN) bacteria cell
proliferates within the gastrointestinal tract and a biofilm
including a population of Nissle strain 1917 (EcN) bacteria cells
is formed that includes the diagnostic polypeptide.
81. The method of claim 80 wherein the diagnostic polypeptide is
detected.
82. The method of claim 80 wherein the diagnostic polypeptide
further comprises an imaging agent or a dye.
83. The method of claim 80 wherein the diagnostic polypeptide is
released.
84. The method of claim 80 wherein the linker is a cleavable linker
and the diagnostic polypeptide is released by cleaving the
linker.
85. The method of claim 80 wherein the linker is a cleavable linker
and the diagnostic polypeptide is released by cleaving the linker
with an enzyme.
86. The method of claim 80 wherein the linker is a cleavable linker
and the diagnostic polypeptide is released by cleaving the linker
with an enzyme within the gastrointestinal tract.
87. A method of binding capture targets within the gastrointestinal
tract of an organism comprising introducing into the organism a
Nissle strain 1917 (EcN) bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
capture agent polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);
wherein the Nissle strain 1917 (EcN) bacteria cell proliferates
within the gastrointestinal tract and a biofilm including a
population of Nissle strain 1917 (EcN) bacteria cells is formed
that includes the capture agent polypeptide, and wherein the
capture target binds to the capture agent polypeptide.
88. The method of claim 87 wherein the biofilm including bound
capture target is excreted from the organism.
89. The method of claim 87 wherein the biofilm including bound
capture target is removed from tissue and is excreted from the
organism.
90. The method of claim 87 wherein the biofilm including bound
capture target is removed from tissue by natural processes and is
excreted from the organism.
91. The method of claim 87 wherein the capture agent polypeptide is
released.
92. The method of claim 87 wherein the linker is a cleavable linker
and the capture agent polypeptide is released by cleaving the
linker.
93. The method of claim 87 wherein the linker is a cleavable linker
and the capture agent polypeptide is released by cleaving the
linker with an enzyme.
94. The method of claim 87 wherein the linker is a cleavable linker
and the capture agent polypeptide is released by cleaving the
linker with an enzyme within the gastrointestinal tract.
95. A method of delivering a functional agent to the
gastrointestinal tract of an organism comprising introducing into
the organism a Nissle strain 1917 (EcN) bacteria cell expressing a
foreign nucleic acid sequence encoding a fusion of a CsgA protein
linked to a polypeptide that is a first member of a binding pair of
molecules by a linker, wherein the linker comprises [GGGS]n wherein
n is an integer from 2 to 20 (SEQ ID NO:12); wherein the Nissle
strain 1917 (EcN) bacteria cell proliferates within the
gastrointestinal tract and a biofilm including a population of
Nissle strain 1917 (EcN) bacteria cells is formed that includes the
first member of a binding pair of molecules, introducing into the
gastrointestinal tract a second member of the binding pair of
molecules having a functional agent attached thereto, wherein the
first and second members of the binding pair of molecules bind to
each other, thereby attaching the functional agent to the
biofilm.
96. A bacteria cell genetically modified to lack a native sequence
encoding a CsgA protein.
97. The bacteria of claim 96 which is E. coli.
98. The bacteria of claim 96 which is Nissle strain 1917 (EcN).
99. A bacteria cell genetically modified to lack a native sequence
encoding a CsgA protein and including a foreign nucleic acid
sequence encoding a fusion of a CsgA protein linked to a non-native
functional polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12).
100. A bacteria cell genetically modified to lack a native sequence
encoding a CsgA protein and including a vector comprising a nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
non-native functional polypeptide by a linker, wherein the linker
comprises [GGGS]n wherein n is an integer from 2 to 20 (SEQ ID
NO:12).
101. A bacteria cell genetically modified to lack a native sequence
encoding a CsgA protein and expressing a foreign nucleic acid
sequence encoding a fusion of a CsgA protein linked to a non-native
functional polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12).
102. A method of treating an organism with inflammation of the
gastrointestinal tract comprising introducing into the organism an
E. coli bacteria cell expressing a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to a tissue binding
polypeptide by a linker, wherein the linker comprises [GGGS]n
wherein n is an integer from 2 to 20 (SEQ ID NO:12); wherein the E.
coli bacteria cell attaches to tissue of the gastrointestinal tract
by the tissue binding polypeptide thereby localizing the E. coli
bacteria cell to the tissue in a manner to reduce the
inflammation.
103. The method of claim 102 wherein the E. coli bacteria cell
proliferates and a biofilm including a population of E. coli
bacteria cells is formed that is attached to the tissue.
104. The method of claim 102 wherein the tissue binding polypeptide
is CP15, P8, A1, T18, TTF1, TTF2 or TTF3.
105. The method of claim 102 wherein the inflammation results from
inflammatory bowel disease.
106. The method of claim 102 wherein the inflammation results from
Crohn's disease or ulcerative colitis.
107. A method of treating an organism with inflammation of the
gastrointestinal tract comprising introducing into the organism an
E. coli bacteria cell expressing a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to an anti-inflammatory
polypeptide by a linker, wherein the linker comprises [GGGS]n
wherein n is an integer from 2 to 20 (SEQ ID NO:12); wherein the E.
coli bacteria cell proliferates within the gastrointestinal tract
and a biofilm including a population of E. coli bacteria cells is
formed that includes the anti-inflammatory polypeptide in a manner
to reduce the inflammation.
108. The method of claim 107 wherein the anti-inflammatory
polypeptide is TTF1, TTF2 or TTF3.
109. The method of claim 107 wherein the inflammation results from
inflammatory bowel disease or Crohn's disease.
110. The method of claim 107 wherein the anti-inflammatory
polypeptide is released.
111. The method of claim 107 wherein the linker is a cleavable
linker and the anti-inflammatory polypeptide is released by
cleaving the linker.
112. The method of claim 107 wherein the linker is a cleavable
linker and the anti-inflammatory polypeptide is released by
cleaving the linker with an enzyme.
113. The method of claim 107 wherein the linker is a cleavable
linker and the anti-inflammatory polypeptide is released by
cleaving the linker with a protease produced as a result of the
inflammation.
114. The method of claim 107 wherein the linker is a cleavable
linker and the anti-inflammatory polypeptide is released by
cleaving the linker with an MMP protease.
115. A method of treating an organism with cancer of the
gastrointestinal tract comprising introducing into the organism an
E. coli bacteria cell expressing a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to an anti-cancer
polypeptide by a linker, wherein the linker comprises [GGGS]n
wherein n is an integer from 2 to 20 (SEQ ID NO:12); wherein the E.
coli bacteria cell proliferates within the gastrointestinal tract
and a biofilm including a population of E. coli bacteria cells is
formed that includes the anti-cancer polypeptide in a manner to
reduce proliferation of the cancer.
116. The method of claim 115 wherein the anti-cancer polypeptide is
a growth inhibiting biologic.
117. The method of claim 115 wherein the anti-cancer polypeptide is
released.
118. The method of claim 115 wherein the linker is a cleavable
linker and the anti-cancer polypeptide is released by cleaving the
linker.
119. The method of claim 115 wherein the linker is a cleavable
linker and the anti-cancer polypeptide is released by cleaving the
linker with an enzyme.
120. The method of claim 115 wherein the linker is a cleavable
linker and the anti-cancer polypeptide is released by cleaving the
linker with an enzyme within the gastrointestinal tract.
121. A method of treating an organism with microbial pathogens in
the gastrointestinal tract comprising introducing into the organism
an E. coli bacteria cell expressing a foreign nucleic acid sequence
encoding a fusion of a CsgA protein linked to an anti-microbial
polypeptide by a linker, wherein the linker comprises [GGGS]n
wherein n is an integer from 2 to 20 (SEQ ID NO:12); wherein the E.
coli bacteria cell proliferates within the gastrointestinal tract
and a biofilm including a population of E. coli bacteria cells is
formed that includes the anti-microbial polypeptide in a manner to
reduce proliferation of the microbial pathogens.
122. The method of claim 121 wherein the microbial pathogen is
Clostridium difficile.
123. The method of claim 121 wherein the anti-microbial polypeptide
is coprisin, thurcin CD, a lanibiotic, nisin, actagardine, a
cathelicidin or LL-37.
124. The method of claim 121 wherein the anti-microbial polypeptide
is released.
125. The method of claim 121 wherein the linker is a cleavable
linker and the anti-microbial polypeptide is released by cleaving
the linker.
126. The method of claim 121 wherein the linker is a cleavable
linker and the anti-microbial polypeptide is released by cleaving
the linker with an enzyme.
127. The method of claim 121 wherein the linker is a cleavable
linker and the anti-microbial polypeptide is released by cleaving
the linker with an enzyme within the gastrointestinal tract.
128. The method of claim 121 wherein the linker is a cleavable
linker and the anti-microbial polypeptide is released by cleaving
the linker with protease CD2830 within the gastrointestinal
tract.
129. The method of claim 121 wherein the linker is a cleavable
linker and the anti-microbial polypeptide is released when exposed
to microbial pathogen.
130. A method of delivering a diagnostic polypeptide to the
gastrointestinal tract of an organism comprising introducing into
the organism an E. coli bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
diagnostic polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);
wherein the E. coli bacteria cell proliferates within the
gastrointestinal tract and a biofilm including a population of E.
coli bacteria cells is formed that includes the diagnostic
polypeptide.
131. The method of claim 130 wherein the diagnostic polypeptide is
detected.
132. The method of claim 130 wherein the diagnostic polypeptide
further comprises an imaging agent or a dye.
133. The method of claim 130 wherein the diagnostic polypeptide is
released.
134. The method of claim 130 wherein the linker is a cleavable
linker and the diagnostic polypeptide is released by cleaving the
linker.
135. The method of claim 130 wherein the linker is a cleavable
linker and the diagnostic polypeptide is released by cleaving the
linker with an enzyme.
136. The method of claim 130 wherein the linker is a cleavable
linker and the diagnostic polypeptide is released by cleaving the
linker with an enzyme within the gastrointestinal tract.
137. A method of binding capture targets within the
gastrointestinal tract of an organism comprising introducing into
the organism an E. coli bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
capture agent polypeptide by a linker, wherein the linker comprises
[GGGS]n wherein n is an integer from 2 to 20 (SEQ ID NO:12);
wherein the E. coli bacteria cell proliferates within the
gastrointestinal tract and a biofilm including a population of E.
coli bacteria cells is formed that includes the capture agent
polypeptide, and wherein the capture target binds to the capture
agent polypeptide.
138. The method of claim 137 wherein the biofilm including bound
capture target is excreted from the organism.
139. The method of claim 137 wherein the biofilm including bound
capture target is removed from tissue and is excreted from the
organism.
140. The method of claim 137 wherein the biofilm including bound
capture target is removed from tissue by natural processes and is
excreted from the organism.
141. The method of claim 137 wherein the capture agent polypeptide
is released.
142. The method of claim 137 wherein the linker is a cleavable
linker and the capture agent polypeptide is released by cleaving
the linker.
143. The method of claim 137 wherein the linker is a cleavable
linker and the capture agent polypeptide is released by cleaving
the linker with an enzyme.
144. The method of claim 137 wherein the linker is a cleavable
linker and the capture agent polypeptide is released by cleaving
the linker with an enzyme within the gastrointestinal tract.
145. A method of delivering a functional agent to the
gastrointestinal tract of an organism comprising introducing into
the organism an E. coli bacteria cell expressing a foreign nucleic
acid sequence encoding a fusion of a CsgA protein linked to a
polypeptide that is a first member of a binding pair of molecules
by a linker, wherein the linker comprises [GGGS]n wherein n is an
integer from 2 to 20 (SEQ ID NO:12); wherein the E. coli bacteria
cell proliferates within the gastrointestinal tract and a biofilm
including a population of E. coli bacteria cells is formed that
includes the first member of a binding pair of molecules,
introducing into the gastrointestinal tract a second member of the
binding pair of molecules having a functional agent attached
thereto, wherein the first and second members of the binding pair
of molecules bind to each other, thereby attaching the functional
agent to the biofilm.
Description
RELATED APPLICATION DATA
[0001] This application claims priority to U.S. Provisional
Application No. 62/143,560, filed on Apr. 6, 2015 which is hereby
incorporated herein by reference in its entirety for all
purposes.
FIELD
[0003] The technology described herein relates to engineered
polypeptides, bacteria comprising such polypeptides, engineered
bacteria, biofilms comprising said bacterial cells, biofilms
comprising the engineered polypeptides produced by the engineered
bacterial cells, and amyloid-based extracellular matrix components
produced by engineered bacterial cells.
BACKGROUND
[0004] In nature, most bacteria exist as biofilm communities,
residing in a self-generated protective nanoscale scaffold of
proteins, sugars, lipids, and extracellular DNA that defends
against environmental rigors. Biofilm formation is essential for
bacterial adhesion and colonization of both natural and man-made
surfaces. These highly evolved extracellular matrices hold untapped
potential as a beneficial nanobiotechnology engineering platform.
Biofilms have been investigated for beneficial purposes such as
wastewater treatment and biotransformations, but these efforts
focus on the use of naturally occurring organisms. Efforts to
engineer the structure of biofilms exist. See WO/2012/166906 and
PCT/US2014/035095.
SUMMARY
[0005] Embodiments of the present disclosure are directed to
methods of genetically modifying bacteria to create amyloid-based
materials, such as biofilms created by amyloid fibers, having
non-native functional polypeptides expressed thereon and connected
thereto by a linker domain optimized for functioning of the
non-native functional polypeptides. According to one aspect, the
linker domain is optimized for functioning of a CsgA protein as is
it assembled into an amyloid state and for functioning of the
functional polypeptide. Exemplary biofilms may include living
bacterial cells, non-living bacterial cells or combinations of
living bacterial cells and non-living bacterial cells.
[0006] Exemplary bacteria as described herein include E. coli.
Exemplary bacteria as described herein include non-pathogenic
bacteria. Exemplary bacteria as described herein include Nissle
strain 1917 (EcN), MG1655, K12-derived strains, such as LSR10 and
PHL628. Exemplary bacteria as described herein include bacteria
that have been genetically modified to remove the nucleic acid
sequence or nucleic acid sequences encoding the CsgA protein, i.e.
the CsgA gene. Exemplary bacteria as described herein include
bacteria that have been genetically modified to include a genomic
deletion of the nucleic acid sequence or nucleic acid sequences
encoding the CsgA protein, i.e., the CsgA gene. Exemplary bacteria
as described herein are useful in the methods also described
herein, such as the therapeutic or diagnostics methods described
herein.
[0007] According to one aspect, a functional polypeptide is linked
to the CsgA protein by a linker to form a CsgA-linker-functional
polypeptide structure. According to one aspect, one or more nucleic
acid sequences which encode for the CsgA-linker-functional
polypeptide structure are included in a bacteria cell or the
bacteria cell is genetically modified to include one or more
nucleic acid sequences which encode for the CsgA-linker-functional
polypeptide structure. The one or more nucleic acid sequences,
which may be foreign nucleic acid sequences, are inserted into the
bacteria using methods known to those of skill in the art and the
bacteria expresses the CsgA-linker-functional polypeptide
structure. According to one aspect, a naturally occurring bacteria
is modified to include one or more foreign nucleic acid sequences
thereby resulting in a non-naturally occurring bacteria. According
to one aspect, the non-naturally occurring CsgA-linker-functional
polypeptide structure is secreted and assembled to produce an
amyloid nanofiber network which includes the functional
polypeptide. In an exemplary embodiment, the functional polypeptide
is on the surface of the amyloid nanofiber network and provides the
amyloid nanofiber network with the property or characteristic of
the functional polypeptide. Functional polypeptides of various
lengths, secondary structures, properties, characteristics,
functions, and the like are envisioned as being attached to the
CsgA protein while still allowing amyloid formation and while
imparting the properties, characteristics, functions of the
functional polypeptide to the biofilm.
[0008] According to one aspect, an engineered bacteria is provided
as is an engineered CsgA protein insofar as the CsgA protein
includes the linker and the functional polypeptide and the
engineered bacteria includes one or more nucleic acid sequences
encoding the CsgA-linker-functional polypeptide structure. The
engineered bacteria may or may not include a genomic deletion of
the natural CsgA gene as described above. According to this aspect,
both the engineered bacteria and the engineered CsgA protein are
non-naturally occurring.
[0009] According to one aspect, the CsgA may have attached thereto
two or more linkers to each of which is attached a functional
polypeptide or functional group. According to one aspect, the
amyloid network may include two or more CsgA species, each attached
to its own linker. According to this aspect, a CsgA protein may
have attached thereto two or more or a plurality of linkers each of
which may or may not have a functional polypeptide or functional
group attached thereto to provide a CsgA protein with a two or more
or a plurality of functional polypeptides or functional groups
attached thereto. Each functional polypeptide or functional group
may be the same or different. For example, one functional
polypeptide or group attached to the CsgA protein via a first
linker may be a cell specific binding functional polypeptide or
functional group and a different functional polypeptide or
functional group attached to a different CsgA via a second linker
may be a therapeutic or diagnostic functional polypeptide or
functional group. According to this aspect, the assembled hybrid
amyloid fiber, composed of the two CsgA variants can target a
particular tissue or cell type and also deliver a therapeutic or
diagnostic agent to the tissue or cell type. Linkers may be
attached at any location of the CsgA protein as is feasible
including the N-terminus, the C-terminus or at locations in between
the N-terminus and the C-terminus. Further, two or more or a
plurality of linker domains may be attached in series with a
functional polypeptide or group attached to each linker, thereby
providing two or more or a plurality of functional polypeptides or
groups to the CsgA protein.
[0010] Accordingly, methods described herein include the
proliferation of the bacteria cell or bacterial cells to create a
population of bacteria cells that expression the foreign nucleic
acid to create curli fibers and a biofilm including the functional
polypeptide.
[0011] Aspects according to the present disclosure include
administering the bacteria as described herein to an organism for
therapeutic or diagnostic purposes. An organism includes a mammal,
such as a human or a non-human mammal.
[0012] Further features and advantages of certain embodiments of
the present invention will become more fully apparent in the
following description of embodiments and drawings thereof, and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee. The foregoing and
other features and advantages of the present embodiments will be
more fully understood from the following detailed description of
illustrative embodiments taken in conjunction with the accompanying
drawings in which:
[0014] FIG. 1 depicts results of a Congo-Red plate assay where the
presence of extracellular amyloid fibers is shown by red staining
of the culture spots. The data was obtained from a LSR10
strain.
[0015] FIG. 2 depicts results of a whole-cell immunodotblot
analysis for FLAG tag functionality. Accessible FLAG epitopes on
CsgA-FLAG chimeras with different linker domains were probed using
anti-FLAG antibodies conjugated to fluorescent DyLight 680. The
data was obtained from a LSR10 strain.
[0016] FIG. 3 depicts in schematic a strategy for optimized linker
design showing the effect of optimized linker on functional
polypeptide ("material") performance. Unoptimized linker domains
may hinder the performance of the biofilm-based material by
occluding binding sites for the functional domains, preventing
proper folding of either domain, hindering assembly of the amyloid
fibers, or hindering secretion. Optimized linkers minimize these
issues, thereby improving the desired function of the material
overall.
[0017] FIG. 4 is a graph depicting expression of curli nanofibers
fused with trefoil factor proteins determined by a CR absorbance
assay for various CsgA-TFF constructs with various length flexible
linker domains. The data was obtained from a PHL628 strain.
[0018] FIG. 5 is a graph depicting expression of curli nanofibers
fused with gut binding short peptide domains using a Congo red
binding assay of wild type CsgA compared to CsgA fused to four
different peptides that have bene shown to bind to specific gut
tissues. The data was obtained from a PHL628 strain.
[0019] FIG. 6 is a graph depicting adhesion of LSR10 cells
harboring gut-binding domains to Caco-2 cells where constructs with
the F48 linker and containing trefoil domains TTF1, TTF2, and TTF3,
or small peptides T18, A1, CP15 and P8 were tested for in vitro
binding to Caco-2 monolayers. Bound cells were recovered and
quantified by CFU analysis.
[0020] FIG. 7 is a graph depicting adhesion of PHL628 cells
producing engineered curli fibers to Caco-2 cell monolayers. When
no curli is produced, adhesion decreases significantly, suggesting
that the wild type curli fibers may play a role in adhesion to
epithelial surfaces. TTF1 and TTF3 increase adhesion to the
epithelial surface.
[0021] FIG. 8A-8D depict construction of a construction of a csgA
deletion mutant of Nissle 1917. FIG. 8A depicts a Lambda red
recombination strategy for the deletion of the csgA gene in Nissle.
FIG. 8B depicts PCR validation of the chloramphenicol cassette
insertion at the csgA locus identifies two positive clones (black
arrows). FIG. 8C depicts these two clones were verified for the
presence of the 265, 158, and 1113 bp amplicons. FIG. 8D depicts
sequencing verification of the regions flanking the csgA gene
indicates successful CAT cassette integration (highlighted in
yellow) (SEQ ID NO:2).
[0022] FIG. 9 is a graph depicting adhesion of Nissle PBP17
harboring gut-binding TFFs to Caco-2 cells. Constructs with the F48
linker and containing trefoil domains (TFF1, TFF2, and TFF3) were
expressed in a Nissle .DELTA.csgA mutant (PBP17) and tested for in
vitro binding to Caco-2 monolayers. Bound cells were recovered and
quantified by CFU analysis and the data is presented as the percent
bound compared to the initial inoculum.
[0023] FIG. 10 depicts a diagram of CsgA-linker-AMP constructs to
be synthesized and screened. Sec is a periplasmic secretion tag
that is cleaved after transport. N22 is the outer membrane
secretion tag. CsgA is the amyloidogenic region of the protein.
Linkers will have the general sequence (GGS).sub.nXXXX, with n=3,
4, or 5 and XXXX=PPP, PPIP, or PPVP (SEQ ID NO:3). The total panel
size will be 9 members, with fused domains ranging from 22-28 amino
acids.
DETAILED DESCRIPTION
[0024] Aspects of the present disclosure are directed to structures
having a self-assembling domain and a variable functional domain
interconnected by a linker domain. According to one aspect, the
self-assembling domain is a bacterial matrix protein. According to
one aspect, the self-assembling domain is an amyloidogenic
protein.
[0025] According to one aspect, the self-assembling domain is the
amyloidogenic protein, CsgA. As used herein, "CsgA" (as
distinguished from an engineered CsgA polypeptide) refers to the
major structural subunit of curli. The sequences of CsgA and its
homologs are known in a number of species, e.g. the sequence of E.
coli CsgA is known (NCBI Gene ID NO: 949055; SEQ ID NO: 1
(polypeptide)).
[0026] CsgA polypeptide NCBI Ref Seq: NP_415560 [0027] 1 mkllkvaaia
aivfsgsala gwpqygggg nhggggnnsg pnselniyqy gggnsalalq [0028] 61
tdarnsdlti tqhgggngad vgqgsddssi dltqrgfgns atldqwngkn semtvkqfgg
[0029] 121 gngaavdqta snssvnvtqv gfgnnatahq y [SEQ ID NO:1].
[0030] In some embodiments, "CsgA" refers to E. coli CsgA. In some
embodiments, "CsgA" refers to a polypeptide having at least 80%
homology to SEQ ID NO: 1 (e.g. 80% or greater homology, 90% or
greater homology, or 95% or greater homology), e.g. naturally
occurring mutations or variants of CsgA, homologs of CsgA, or
engineered mutations or variants of CsgA. As used herein, an
"engineered CsgA polypeptide" refers to a CsgA polypeptide
comprising a linker and a functional polypeptide attached to the
CsgA at either the C-terminus or the N-terminus or both, but
without interrupting the sequence of the CsgA polypeptide.
[0031] According to one aspect, structures according to the present
disclosure include a CsgA protein, a linker and a functional
polypeptide. According to one aspect, a CsgA protein is attached to
a functional polypeptide by a linker. According to one aspect, the
functional polypeptide is a heterologous peptide or protein domain.
According to one aspect, the functional polypeptide is a
heterologous peptide or protein domain that is foreign to the
bacterial cell which will express the heterologous peptide or
protein domain. According to one aspect, the CsgA protein, linker
and functional polypeptide are attached in series, for example,
having the structure CsgA-linker-functional polypeptide. According
to one aspect, the CsgA protein and the functional polypeptide each
exhibit functionality while bound together through the linker.
According to one aspect, the combined length of the linker and
functional polypeptide can be within the range of 10-500 amino
acids, 10-450 amino acids, 10-400 amino acids, 10-350 amino acids
or 10-300 amino acids.
[0032] According to one aspect, bacteria are modified to include a
nucleic acid encoding the CsgA-linker-functional polypeptide
structure. Methods of introducing a nucleic acid to a bacteria cell
are known to those of skill in the art. The modified bacteria
secrete the CsgA-linker-functional polypeptide structure which
results in curli fiber production followed by biofilm formation.
The CsgA, linker and functional protein structure are produced by
engineered or non-naturally occurring bacteria and the CsgA and
functional protein exhibit proper folding and exhibit
functionality. According to one aspect, methods are provided for
engineering a bacteria to produce a CsgA-linker-functional
polypeptide structure which is exported from the bacteria and
assembled into extracellular amyloid fibers. After secretion, the
CsgA is nucleated to form an amyloid at the cell surface, and then
continues to polymerize into long fibers that eventually
encapsulate the cells and provide the biofilm with structural
support. Attached to each CsgA is the linker and the functional
polypeptide as a fusion. The structure is secreted and the
functional polypeptide is displayed on the surface of the
extracellular amyloid network. The domains are chosen such that
they may have functions that can alter or enhance the properties of
the biofilm as a whole and the linkers are chosen to allow the
domains to have their particular functions.
[0033] Functional polypeptides within the scope of the present
disclosure include peptides or proteins having a desired function.
Such functions include catalytic function, recognition function or
structural function. Exemplary functional polypeptides include
targeting domains. Exemplary functional polypeptides include
therapeutic polypeptides. Exemplary functional polypeptides include
diagnostic polypeptides. Exemplary functional polypeptides include
anticancer polypeptides. Exemplary functional polypeptides include
antimicrobial polypeptides. Exemplary functional polypeptides
include anti-inflammatory polypeptides. Exemplary functional
polypeptides include polymer binding polypeptides. Exemplary
functional polypeptides include metabolite binding polypeptides.
Exemplary functional polypeptides include targeting polypeptides.
Exemplary functional polypeptides include functional polypeptides
that bind to tissues or cells or substrates. For example, by
appending a domain with known steel binding capabilities to CsgA, a
biofilm is produced with the ability to adhere to steel surfaces,
whereas the wild-type biofilm does not have this capability.
Exemplary functional polypeptides include a first member of a known
binding pair. When expressed, the first member of the binding pair
is available for binding to a second member of the binding pair
which may have attached to it a functional polypeptide, such as for
therapeutic or diagnostic purposes. In this manner, the functional
polypeptide with the second member of the binding pair may be
contacted to the biofilm to add the functional polypeptide to the
biofilm, such as to provide the biofilm with the characteristic of
the functional polypeptide. Exemplary functional polypeptides may
be those to which a functional group may be covalently attached
either directly or through a linker. For example, by appending to
CsgA a peptide capable of undergoing spontaneous covalent
modification, a biofilm whose surface can be modified with any
protein or compound of interest can be created by subsequent
addition of the protein or compound of interest.
[0034] Exemplary therapeutic polypeptides include engineered
polypeptides with therapeutic function, polypeptides with
anti-inflammatory bioactivity (trefoil factors--e.g. TFF1-3,
interleukins--e.g. IL-10, other anti-inflammatory cytokines,
anti-TNF.alpha. factors), polypeptides with anti-microbial
bioactivity (e.g. coprisin, cathelicidin, LL-37, thuricin CD,
lantibiotics), polypeptides with anti-cancer bioactivity (growth
inhibiting biologics).
[0035] Exemplary diagnostic polypeptides include those known to
those of skill in the art and identified by literature search.
[0036] Exemplary anticancer polypeptides include polypeptides with
anti-cancer bioactivity (growth inhibiting biologics) and
anticancer polypeptides include those known to those of skill in
the art and identified by literature search.
[0037] Exemplary antimicrobial polypeptides include coprisin,
cathelicidin, LL-37, thuricin CD, lantibiotics and antimicrobial
polypeptides known to those of skill in the art and identified by
literature search.
[0038] Exemplary anti-inflammatory polypeptides include trefoil
factors--e.g. TFF1-3, interleukins--e.g. IL-10, other
anti-inflammatory cytokines, anti-TNF.alpha. factors and
anti-inflammatory polypeptides known to those of skill in the art
and identified by literature search.
[0039] Exemplary polymer binding polypeptides include those known
to those of skill in the art and identified by literature
search.
[0040] Exemplary metabolite binding polypeptides include those
known to those of skill in the art and identified by literature
search.
[0041] Exemplary targeting polypeptides include those known to
those of skill in the art and identified by literature search.
[0042] Exemplary tissue-binding polypeptides include T18, CP15 and
those known to those of skill in the art and identified by
literature search.
[0043] Exemplary cell-binding polypeptides include T18, CP15 and
those known to those of skill in the art and identified by
literature search.
[0044] Exemplary polypeptides that are a first pair of a binding
pair of molecules include coiled-coil domains such as SynZips,
Trp-Zip domains, affinity tags such as FLAG, and the like and those
known to those of skill in the art and identified by literature
search
[0045] Linkers within the scope of the present disclosure are
characterized in terms of amino acid content, length, rigidity and
secondary structure. Linkers within the scope of the present
disclosure separate the amyloid domain and the functional
polypeptide domain and allow proper folding and functioning of each
domain. In this manner, a linker can be tailored to the particular
amyloid domain and the particular functional polypeptide domain.
According to one aspect, functional independence of the structural
(i.e., CsgA) and fused (heterologous) domains is maximized by a
suitable linker to limit steric interference between domains during
the export and assembly processes of the bacterial cell. According
to one aspect, longer and more flexible linkers of the type
(GGGS).sub.n (SEQ ID NO:4) are exemplary. According to an
additional aspect, cell stress is minimized by limiting the overall
length of the fusion protein. Longer linker sequences and higher
induction levels stress the biosynthetic machinery of the cells,
inhibiting cell growth and leading to cell lysis in extreme
cases.
[0046] Linkers within the scope of the present disclosure
facilitate functioning of the CsgA domain and the functional
peptide domain. Linkers within the scope of the present disclosure
allow efficient protein processing and export through the bacterial
curli secretion machinery as well as provide the proper spatial and
physicochemical separation of the amyloid and functional domains to
retain their respective functions.
[0047] Linkers within the scope of the present disclosure include
amino acid residues. The amino acid residues may be any of the
naturally occurring amino acid residues. Amino acid residues may
also be synthetic amino acids known to those of skill in the art.
Representative amino acids which may be used in linkers include
Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Cysteine,
Selenocysteine, Threonine, Methionine, Proline, Phenylalanine,
Tyrosine, Tryptophan, Histidine, Lysine, Arginine, Aspartate,
Glutamate, Asparagine, and Glutamine.
[0048] Linkers within the scope of the present disclosure include a
cleavage site. Cleavage sites and enzymes for cleavage are known to
those of skill in the art. According to this embodiment, the
functional polypeptide may be cleaved from the linker and released
into the surrounding environment, for example for therapeutic or
diagnostic purposes. Exemplary enzymes include those from the
family of matrix metalloproteinases (MMPs), which have their own
recognition sequences, proteases secreted by pathogens such as
CD2830 from C. difficile and the like.
[0049] CsgA within the scope of the present disclosure includes an
amyloid domain which self-assembles into an amyloid structure.
According to certain aspects, a linker may be attached to either
the C terminus or the N-terminus or separate linkers may be
attached to both the C terminus and the N terminus.
[0050] According to one aspect, the linker length can be any length
which may be expressed from a cell, such as a bacterial cell when
linking a CsgA protein and a functional polypeptide. According to
one aspect, the functional polypeptide length can be any length
which may be expressed from a cell, such as a bacterial cell when
linked to a CsgA protein by a linker. According to one aspect, the
combination of a linker and functional polypeptide includes no more
than 500 amino acids. In some embodiments, the combination of a
linker and functional polypeptide includes no more than 400 amino
acids. In some embodiments, the combination of a linker and
functional polypeptide includes no more than 300 amino acids. In
some embodiments, the combination of a linker and functional
polypeptide includes no more than 200 amino acids. In some
embodiments, the combination of a linker and functional polypeptide
includes no more than 100 amino acids. In some embodiments, the
combination of a linker and functional polypeptide includes no more
than 50 amino acids. In some embodiments, the combination of a
linker and functional polypeptide includes no more than 40 amino
acids. In some embodiments, the combination of a linker and
functional polypeptide includes no more than 30 amino acids.
[0051] According to one aspect, a linker sequence is a polypeptide
sequence of at least 7, 8, 9, 10, 11, 12, 24, 48 or more amino
acids. In some embodiments, the linker sequence comprises from
about 7 amino acids to about 250 amino acids. In some embodiments,
the linker sequence comprises from about 7 amino acids to about 200
amino acids. In some embodiments, the linker sequence comprises
from about 7 amino acids to about 150 amino acids. In some
embodiments, the linker sequence comprises from about 7 amino acids
to about 100 amino acids. In some embodiments, the linker sequence
comprises from about 12 amino acids to about 250 amino acids. In
some embodiments, the linker sequence comprises from about 12 amino
acids to about 200 amino acids. In some embodiments, the linker
sequence comprises from about 12 amino acids to about 150 amino
acids. In some embodiments, the linker sequence comprises from
about 12 amino acids to about 100 amino acids. In some embodiments,
the linker sequence comprises from about 24 amino acids to about
100 amino acids. In some embodiments, the linker sequence comprises
from about 48 amino acids to about 250 amino acids. In some
embodiments, the linker sequence comprises from about 48 amino
acids to about 200 amino acids. In some embodiments, the linker
sequence comprises from about 48 amino acids to about 150 amino
acids. In some embodiments, the linker sequence comprises from
about 48 amino acids to about 100 amino acids. In some embodiments,
the linker sequence comprises from about 7 amino acids to about 30
amino acids. In some embodiments, the linker sequence comprises
from about 20 amino acids to about 50 amino acids. In some
embodiments, the linker sequence comprises from about 30 amino
acids to about 50 amino acids. In some embodiments, the linker
sequence comprises from about 40 amino acids to about 50 amino
acids. In some embodiments, the linker sequence comprises from
about 6 amino acids to about 20 amino acids. In some embodiments,
the linker sequence comprises from about 7 to about 10 amino acids.
In some embodiments, the linker sequence comprises a flexible
polypeptide, e.g a polypeptide not having a rigid secondary and/or
tertiary structure. In some embodiments, the linker sequence
comprises glycine and serine residues. In some embodiments at least
50% of the amino acids comprised by the linker sequence are glycine
or serine residues, e.g. at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, or more are glycine or
serine residues. In some embodiments, the linker sequence consists
of glycine and serine residues.
[0052] A functional polypeptide includes a polypeptide having an
activity or function, such that when it is present in a biofilm, it
confers upon the biofilm a property, function, or activity which it
did not have in the absence of the activity of the polypeptide.
Accordingly, an activity polypeptide can be, e.g. an enzyme, a
polypeptide that binds another molecule, a binding domain, a
peptide that is bound by another molecule (e.g. a ligand or
epitope), or the like. Examples of polypeptides for use as activity
polypeptides include, but are not limited to Metal binding domain
(MBD); SpyTag; graphene binding (GBP); carbon nanotube binding
(CBP); gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and
AFP8.
[0053] According to certain aspects, the functional polypeptide
when present as part of an engineered CsgA polypeptide, is
functional. A polypeptide is said to be "functional" or expressed
as a "functional" polypeptide if the polypeptide retains at least
about 50% of the activity (e.g. enzymatic activity or binding
activity) that it has as an isolated polypeptide. One of skill in
the art can readily detect increases in reaction products and/or
detect decreases in reaction substrates, e.g. by mass spectroscopy
(MS, including, e.g., MADLI/TOF, SELDI/TOF, LC-MS, GC-MS, HPLC-MS,
etc., among others) or detect increases or decrease in binding to a
binding partner, e.g. by immunoassays. In some embodiments, a
functional activity polypeptide can retain at least 50% of the
activity of the isolated polypeptide, e.g. 50% or more of the
activity, 60% or more of the activity, 75% or more of the activity,
or 90% or more of the activity of the isolated polypeptide.
[0054] In some embodiments, the functional polypeptide can be a
conjugation domain. Such embodiments can permit immobilization of
target proteins in the biofilm, e.g., when the target protein is
too large to be expressed as a fusion with CsgA. The conjugation
domain present on the engineered CsgA polypeptide can specifically
bind to a partner conjugation domain present as part of the target
protein, thereby incorporating the target protein into the biofilm.
Such conjugation domains are also referred to herein as a first
member of a binding pair of molecules and a second member of a pair
of binding pair of molecules. As used herein, "conjugation domain"
includes a polypeptide that can specifically bind to and/or be
specifically bound by a partner conjugation domain, e.g. under
conditions suitable for growth of a biofilm. A conjugation domain
can be, e.g., about 100 amino acids or less in size, about 75 amino
acids or less in size, about 50 amino acids or less in size, about
40 amino acids or less in size or smaller. A partner conjugation
domain can be about the same size as the conjugation domain or
larger, e.g., a partner conjugation domain can be about 4000 amino
acids or less in size, about 3000 amino acids or less in size,
about 2000 amino acids or less in size, about 1000 amino acids or
less in size, about 500 amino acids or less in size, about 200
amino acids or less in size, about 100 amino acids or less in size,
about 75 amino acids or less in size, about 50 amino acids or less
in size, about 40 amino acids or less in size, or smaller. In some
embodiments, the binding of the conjugation domain and partner
conjugation domain is covalent. Examples of conjugation domains are
known in the art and include, but are not limited to, SpyTag;
biotin acceptor peptide (BAP); biotin carboxyl carrier protein
(BCCP); and a peptide comprising a LPXTG (SEQ ID NO:30) motif.
Similarly, partner conjugation domains are known in the art and
include but are not limited to, respectively, SpyCatcher,
streptavidin; streptavidin; and peptides comprising aminoglycine.
Further discussion of conjugation systems comprising a conjugation
domain and a partner conjugation domain can be found, e.g., in Mao
et al. J Am Chem Soc 2004 126:2670-1; Zakeri et al. PNAS 2012
109:E690-E697; and Maeda et al. Appl Environ Microbil 2008
74:5139-5145; each of which is incorporated by reference herein in
its entirety.
[0055] Where the functional polypeptide is a conjugation domain,
the target polypeptide comprising the partner conjugation domain
can further comprise a functional agent. The functional agent has
an activity or function, such that when it is present in a biofilm,
it confers upon the biofilm a property, function, or activity which
it did not have in the absence of the polypeptide. A functional
agent can be of any size and is not part of the engineered CsgA
polypeptide. Exemplary functional agents include, e.g. an enzyme, a
polypeptide that binds another molecule, an antibody, a therapeutic
agent, a diagnostic agent, a metal, an antimicrobial agent, an
anti-inflammatory agent, an anticancer agent or the like. In some
embodiments, a polypeptide comprising a functionalizing polypeptide
and a conjugation domain can further comprise an extracellular
localization tag, e.g. a sequence which will cause a cell
expressing the polypeptide to secrete the polypeptide.
[0056] A functionalized engineered CsgA polypeptide or
functionalized biofilm can be provided by contacting an engineered
CsgA polypeptide comprising a conjugation domain (or a cell and/or
biofilm comprising that polypeptide) with a polypeptide comprising
the partner conjugation domain. In some embodiments, the engineered
CsgA polypeptide and the polypeptide comprising the partner
conjugation domain are maintained in contact for a period of time,
i.e. the "binding step." In some embodiments, the binding step is
followed by a washing step, e.g. to remove excess unbound
polypeptide.
[0057] In some embodiments, an engineered CsgA polypeptide
comprising a conjugation domain is bound to (or binds) the partner
conjugation domain in the presence of albumin (i.e. the "binding
step"). In some embodiments, the albumin is BSA. In some
embodiments, the albumin is present at about 0.1% to about 10%. In
some embodiments, the albumin is present at about 0.5% to about 5%.
In some embodiments, the albumin is present at about 1% to about
2%. In some embodiments, the binding step is allowed to proceed for
at least about 2 hours, e.g. about 2 hours or more, about 6 hours
or more, about 12 hours or more, or about 24 hours or more. In some
embodiments, the binding step is allowed to proceed in the presence
of albumin.
[0058] In some embodiments, the washing step proceeds for about 10
minutes to about 6 hours. In some embodiments, the washing step
proceeds for about 30 minutes to about 3 hours. In some
embodiments, the washing step proceeds for about 90 minutes. In
some embodiments, the polypeptides are agitated (e.g. shaken)
during the washing step. In some embodiments, the washing step
comprises washing the polypeptides in a solution of albumin. In
some embodiments, the albumin is BSA. In some embodiments, the
albumin is present at about 0.01% to about 3%. In some embodiments,
the albumin is present at about 0.1% to about 1%. In some
embodiments, the albumin is present at about 0.3%. In some
embodiments, the washing step comprises 2 or more successive
washes. In some embodiments, the washing step comprises 3
successive washes.
[0059] "Specific binding" includes a chemical interaction between
two molecules, compounds, cells and/or particles wherein the first
entity binds to the second, target entity with greater specificity
and affinity than it binds to a third entity which is a non-target.
In some embodiments, specific binding can refer to an affinity of
the first entity for the second target entity which is at least 10
times, at least 50 times, at least 100 times, at least 500 times,
at least 1000 times or greater than the affinity for the third
nontarget entity. A reagent specific for a given target is one that
exhibits specific binding for that target under the conditions of
the assay being utilized.
[0060] In one aspect, described herein is a nucleic acid sequence
encoding an engineered CsgA polypeptide as described herein. In one
aspect, described herein is a vector comprising a nucleic acid
sequence encoding an engineered CsgA polypeptide as described
herein. A "vector" includes a nucleic acid construct designed for
delivery to a host cell or transfer between different host cells. A
vector can be viral or non-viral. Many vectors useful for
transferring genes into target cells are available, e.g. the
vectors may be episomal, e.g., plasmids, virus derived vectors or
may be integrated into the target cell genome, through homologous
recombination or random integration. In some embodiments, a vector
can be an expression vector. An "expression vector" can be a vector
that has the ability to incorporate and express heterologous
nucleic acid fragments in a cell. An expression vector may comprise
additional elements, for example, the expression vector may have
two replication systems, thus allowing it to be maintained in two
organisms. The nucleic acid incorporated into the vector can be
operatively linked to an expression control sequence when the
expression control sequence controls and regulates the
transcription and translation of that polynucleotide sequence.
[0061] In some embodiments, a nucleic acid encoding an engineered
CsgA polypeptide can be present within a portion of a plasmid.
Plasmid vectors can include, but are not limited to, pBR322,
pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339,
pR290, pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see
"Stratagene Cloning Systems" Catalog (1993) from Stratagene, La
Jolla, Calif, which is hereby incorporated by reference), pQE,
pIH821, pGEX, pET series (see Studier et. al., "Use of T7 RNA
Polymerase to Direct Expression of Cloned Genes," Gene Expression
Technology, vol. 185 (1990), which is hereby incorporated by
reference in its entirety).
[0062] A "viral vector" may be a nucleic acid vector construct that
includes at least one element of viral origin and has the capacity
to be packaged into a viral vector particle. The viral vector can
contain a transgenic gene in place of non-essential viral genes.
The vector and/or particle may be utilized for the purpose of
transferring any nucleic acids into cells either in vitro or in
vivo. Numerous viral vectors are known in the art and can be used
as carriers of a nucleic acid into a cell, e.g. lambda vector
system gill, gt WES.tB, Charon 4.
[0063] In some embodiments, the nucleic acid encoding an engineered
CsgA polypeptide can be constitutively expressed. In some
embodiments, the nucleic acid encoding an engineered CsgA
polypeptide can be operably linked to a constitutive promoter. In
some embodiments, the nucleic acid encoding an engineered CsgA
polypeptide can be inducibly expressed. In some embodiments, the
nucleic acid encoding an engineered CsgA polypeptide can be
operably linked to an inducible promoter. In some embodiments, the
nucleic acid encoding an engineered CsgA polypeptide can be
operably linked to a native CsgA promoter.
[0064] An "inducible promoter" may be one that is characterized by
initiating or enhancing transcriptional activity when in the
presence of, influenced by, or contacted by an inducer or inducing
agent than when not in the presence of, under the influence of, or
in contact with the inducer or inducing agent. An "inducer" or
"inducing agent" may be endogenous, or a normally exogenous
compound or protein that is administered in such a way as to be
active in inducing transcriptional activity from the inducible
promoter. In some embodiments, the inducer or inducing agent, e.g.,
a chemical, a compound or a protein, can itself be the result of
transcription or expression of a nucleic acid sequence (e.g., an
inducer can be a transcriptional repressor protein), which itself
may be under the control or an inducible promoter. Non-limiting
examples of inducible promoters include but are not limited to, the
lac operon promoter, a nitrogen-sensitive promoter, an
IPTG-inducible promoter, a salt-inducible promoter, and
tetracycline, steroid-responsive promoters, rapamycin responsive
promoters and the like. Inducible promoters for use in prokaryotic
systems are well known in the art, see, e.g. the beta.-lactamase
and lactose promoter systems (Chang et al., Nature, 275: 615 (1978,
which is incorporated herein by reference); Goeddel et al., Nature,
281: 544 (1979), which is incorporated herein by reference), the
arabinose promoter system, including the araBAD promoter (Guzman et
al., J. Bacteriol., 174: 7716-7728 (1992), which is incorporated
herein by reference; Guzman et al., J. Bacteriol., 177: 4121-4130
(1995), which is incorporated herein by reference; Siegele and Hu,
Proc. Natl. Acad. Sci. USA, 94: 8168-8172 (1997), which is
incorporated herein by reference), the rhamnose promoter (Haldimann
et al., J. Bacteriol., 180: 1277-1286 (1998), which is incorporated
herein by reference), the alkaline phosphatase promoter, a
tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:
4057 (1980), which is incorporated herein by reference), the
PLtetO-1 and Plac/are-1 promoters (Lutz and Bujard, Nucleic Acids
Res., 25: 1203-1210 (1997), which is incorporated herein by
reference), and hybrid promoters such as the tac promoter. deBoer
et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is
incorporated herein by reference.
[0065] An inducible promoter useful in the methods and systems as
disclosed herein can be induced by one or more physiological
conditions, such as changes in pH, temperature, radiation, osmotic
pressure, saline gradients, cell surface binding, and the
concentration of one or more extrinsic or intrinsic inducing
agents. The extrinsic inducer or inducing agent may comprise amino
acids and amino acid analogs, saccharides and polysaccharides,
nucleic acids, protein transcriptional activators and repressors,
cytokines, toxins, petroleum-based compounds, metal containing
compounds, salts, ions, enzyme substrate analogs, hormones, and
combinations thereof. In specific embodiments, the inducible
promoter is activated or repressed in response to a change of an
environmental condition, such as the change in concentration of a
chemical, metal, temperature, radiation, nutrient or change in pH.
Thus, an inducible promoter useful in the methods and systems as
disclosed herein can be a phage inducible promoter, nutrient
inducible promoter, temperature inducible promoter, radiation
inducible promoter, metal inducible promoter, hormone inducible
promoter, steroid inducible promoter, and/or hybrids and
combinations thereof. Appropriate environmental inducers can
include, but are not limited to, exposure to heat (i.e., thermal
pulses or constant heat exposure), various steroidal compounds,
divalent cations (including Cu2+ and Zn2+), galactose,
tetracycline, IPTG (isopropyl-.beta.-D thiogalactoside), as well as
other naturally occurring and synthetic inducing agents and
gratuitous inducers.
[0066] Inducible promoters useful in the methods and systems as
disclosed herein also include those that are repressed by
"transcriptional repressors" that are subject to inactivation by
the action of environmental, external agents, or the product of
another gene. Such inducible promoters may also be termed
"repressible promoters" where it is required to distinguish between
other types of promoters in a given module or component of the
biological switch converters described herein. Preferred repressors
for use in the present invention are sensitive to inactivation by
physiologically benign agent. Thus, where a lac repressor protein
is used to control the expression of a promoter sequence that has
been engineered to contain a lacO operator sequence, treatment of
the host cell with IPTG will cause the dissociation of the lac
repressor from the engineered promoter containing a lacO operator
sequence and allow transcription to occur. Similarly, where a tet
repressor is used to control the expression of a promoter sequence
that has been engineered to contain a tetO operator sequence,
treatment of the host cell with tetracycline will cause the
dissociation of the tet repressor from the engineered promoter and
allow transcription of the sequence downstream of the engineered
promoter to occur.
[0067] In one aspect, described herein is an engineered microbial
cell comprising an engineered CsgA polypeptide and/or comprising a
vector or nucleic acid encoding such a polypeptide.
[0068] In some embodiments, the engineered CsgA polypeptide can
comprise a functional polypeptide comprising a conjugation domain.
In some embodiments, a cell encoding and/or comprising an
engineered CsgA polypeptide can comprise an activity polypeptide
comprising a conjugation domain can further encode and/or comprise
a second engineered polypeptide comprising a partner conjugation
domain and a functionalizing polypeptide. In some embodiments,
described herein is a population of cells comprising two cell
types, the first cell type encoding and/or comprising an engineered
CsgA polypeptide comprising an activity polypeptide comprising a
conjugation domain and the second cell type encoding and/or
comprising a second engineered polypeptide comprising a partner
conjugation domain and a functionalizing polypeptide. That is, it
is contemplated herein that a single cell can comprise a CsgA
polypeptide with a conjugation domain and also comprise the
polypeptide which will bind to and/or be bound by that CsgA
polypeptide or that a first cell can comprise a CsgA polypeptide
with a conjugation domain and a second cell can comprise the
polypeptide which will bind to and/or be bound by that CsgA
polypeptide. It is further contemplated that an engineered CsgA
polypeptide with a conjugation domain can be contacted with a
second polypeptide comprising a partner conjugation domain and a
functionalizing polypeptide, e.g. the second polypeptide can be
produced (e.g. by a bacteria or eukaryotic cell) and/or synthesized
(and optionally isolated or purified) and then brought in contact
with the engineered CsgA polypeptide, e.g. when the CsgA
polypeptide is present on a cell surface and/or present in a
biofilm.
[0069] A bacterial cell of the methods and compositions described
herein can be any of any species. Preferably, the bacterial cells
are of a species and/or strain which is amenable to culture and
genetic manipulation. In some embodiments, the bacterial cell can
be a gram-positive bacterial cell. In some embodiments, the
bacterial cell can be a gram-negative bacterial cell. In some
embodiments, the parental strain of the bacterial cell of the
technology described herein can be a strain optimized for protein
expression. Non-limiting examples of bacterial species and strains
suitable for use in the present technologies include Escherichia
coli, E. coli BL21, E. coli Tuner, E. coli Rosetta, E. coli JM101,
and derivatives of any of the foregoing. Bacterial strains for
protein expression are commercially available, e.g. EXPRESS.TM.
Competent E. coli (Cat. No. C2523; New England Biosciences;
Ipswich, Mass.). In some embodiments, the cell is an E. coli
cell.
[0070] In some embodiments, the nucleic acid encoding an engineered
CsgA polypeptide is comprised by a cell expressing wild-type CsgA.
In some embodiments, the nucleic acid encoding an engineered CsgA
polypeptide is comprised by a cell with a mutation and/or deletion
of the wild-type CsgA gene, e.g. such that the cell does not
express wild-type CsgA. In some embodiments, the nucleic acid
encoding an engineered CsgA polypeptide is introduced into a cell
by homologous recombination, e.g. such that the nucleic acid
encoding an engineered CsgA polypeptide replaces the wild-type CsgA
gene in the cell.
[0071] In one aspect, described herein is a biofilm comprising an
engineered microbial cell comprising one or more engineered CsgA
polypeptide and/or comprising a vector or nucleic acid encoding
such a polypeptides. As used herein, a "biofilm" refers to a mass
of microorganisms which can adhere or is adhering to a surface. A
biofilm comprises a matrix of extracellular polymeric substances,
including, but not limited to extracellular DNA, proteins,
glyopeptides, and polysaccharides. The nature of a biofilm, such as
its structure and composition, can depend on the particular species
of bacteria present in the biofilm. Bacteria present in a biofilm
are commonly genetically or phenotypically different than
corresponding bacteria not in a biofilm, such as isolated bacteria
or bacteria in a colony.
[0072] In some embodiments, the technology described herein relates
to a biofilm that is produced by culturing an engineered microbial
cell comprising an engineered CsgA polypeptide (and/or comprising a
vector or nucleic acid encoding such a polypeptide) under
conditions suitable for the production of a biofilm. Conditions
suitable for the production of a biofilm can include, but are not
limited to, conditions under which the microbial cell is capable of
logarithmic growth and/or polypeptide synthesis. Conditions may
vary depending upon the species and strain of microbial cell
selected. Conditions for the culture of microbial cells are well
known in the art. Biofilm production can also be induced and/or
enhanced by methods well known in the art, e.g. contacting cells
with subinhibitory concentrations of beta-lactam or aminoglycoside
antibiotics, exposing cells to fluid flow, contacting cells with
exogenous poly-N-acetylglucosamine (PNAG), or contacting cells with
quorum sensing signal molecules. In some embodiments, conditions
suitable for the production of a biofilm can also include
conditions which increase the expression and secretion of CsgA,
e.g. by exogenously expressing CsgD.
[0073] In some embodiments, the biofilm can comprise the cell which
produced the biofilm. In some embodiments, described herein is a
composition comprising an engineered CsgA polypeptide as described
herein.
[0074] When expressed by a cell capable of forming curli, e.g. a
cell expressing CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG or
some subset thereof, CsgA units will be assembled to form curli
filaments, e.g. polymeric chains of CsgA. In some embodiments,
filaments of the polypeptide can be present in the composition. In
some embodiments, the filaments can be part of a proteinaceous
network, e.g. multiple filaments which can be, e.g. interwoven,
overlapping, and/or in contact with each other. In some
embodiments, the proteinaceous network can comprise additional
biofilm components, e.g. materials typically found in an E. coli
biofilm. Non-limiting examples of biofilm components can include
biofilm proteins (e.g. FimA, FimH, Ag43, AidA, and/or TibA) and/or
non-proteinaceous biofilm components (e.g. cellulose, PGA and/or
colonic acid). In some embodiments, the composition can further
comprise an engineered microbial cell comprising an engineered CsgA
polypeptide and/or comprising a vector or nucleic acid encoding
such a polypeptide.
[0075] In one aspect, described herein is the use of a cell,
composition, or biofilm comprising an engineered CsgA polypeptide
(and/or comprising a vector or nucleic acid encoding such a
polypeptide) to display a polypeptide, e.g. within the biofilm,
within the composition, and/or on the cell surface. As used herein,
"display" refers to expressing the polypeptide (e.g. as an activity
polypeptide) in such a manner that it can come in contact with the
extracellular environment. A displayed polypeptide can be capable
of binding with a binding partner, catalyzing an enzymatic
reaction, and/or performing any other activity which it would
perform as an isolated polypeptide.
[0076] It is contemplated herein that a polypeptide displayed
within a biofilm (e.g. an activity polypeptide and/or
functionalizing polypeptide) will retain more activity than a
soluble version of that polypeptide. It is contemplated herein that
a polypeptide displayed within a biofilm (e.g. an activity
polypeptide and/or functionalizing polypeptide) will retain more
activity than a soluble version of that polypeptide when exposed to
activity degrading conditions such as, e.g., high or low pH,
organic solvents, desiccation, high or low temperature, radiation,
etc.
[0077] In one aspect, described herein is the use of a cell,
composition, or biofilm comprising an engineered CsgA polypeptide
(and/or comprising a vector or nucleic acid encoding such a
polypeptide), in an application selected from the group consisting
of biocatalysis; industrial biocatalysis; immobilized biocatalysis;
chemical production; filtration; isolation of molecules from an
aqueous solution; water filtration; bioremediation; nanoparticle
synthesis; nanowire synthesis; display of optically active
materials; biosensors; surface coating; therapeutic biomaterial;
biological scaffold; structural reinforcement of an object; and as
a delivery system for therapeutic agents. Exemplary, non-limiting
embodiments of such applications and specific activity polypeptides
for use therein are described in the Examples herein.
[0078] It is contemplated herein that a cell, composition and/or
biofilm can comprise multiple different engineered CsgA
polypeptides, each of which comprises a different activity
polypeptide, e.g. an engineered CsgA polypeptide comprising an
enzymatic activity polypeptide and an engineered CsgA polypeptide
comprising a binding domain activity polypeptide. A cell,
composition, and/or biofilm can comprise 1 or more engineered CsgA
polypeptides, e.g. 1, 2, 3, 4, 5, 6, or more engineered CsgA
polypeptides.
[0079] The terms "increased", "increase", "enhance", or "activate"
are all used herein to mean an increase by a statically significant
amount. In some embodiments, the terms "increased", "increase",
"enhance", or "activate" can mean an increase of at least 10% as
compared to a reference level, for example an increase of at least
about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 60%, or at least about 70%, or
at least about 80%, or at least about 90% or up to and including a
100% increase or any increase between 10-100% as compared to a
reference level, or at least about a 2-fold, or at least about a
3-fold, or at least about a 4-fold, or at least about a 5-fold or
at least about a 10-fold increase, or any increase between 2-fold
and 10-fold or greater as compared to a reference level.
[0080] As used herein, the terms "protein" and "polypeptide" are
used interchangeably herein to designate a series of amino acid
residues, connected to each other by peptide bonds between the
alpha-amino and carboxy groups of adjacent residues. The terms
"protein", and "polypeptide" refer to a polymer of amino acids,
including modified amino acids (e.g., phosphorylated, glycated,
glycosylated, etc.) and amino acid analogs, regardless of its size
or function. "Protein" and "polypeptide" are often used in
reference to relatively large polypeptides, whereas the term
"peptide" is often used in reference to small polypeptides, but
usage of these terms in the art overlaps. The terms "protein" and
"polypeptide" are used interchangeably herein when referring to a
gene product and fragments thereof. Thus, exemplary polypeptides or
proteins include gene products, naturally occurring proteins,
homologs, orthologs, paralogs, fragments and other equivalents,
variants, fragments, and analogs of the foregoing.
[0081] A "nucleic acid" or "nucleic acid sequence" may be any
molecule, preferably a polymeric molecule, incorporating units of
ribonucleic acid, deoxyribonucleic acid or an analog thereof. The
nucleic acid can be either single-stranded or double-stranded. A
single-stranded nucleic acid can be one nucleic acid strand of a
denatured double-stranded DNA. Alternatively, it can be a
single-stranded nucleic acid not derived from any double-stranded
DNA. In one aspect, the nucleic acid can be DNA. In another aspect,
the nucleic acid can be RNA. Suitable nucleic acid molecules are
DNA, including genomic DNA or cDNA. Other suitable nucleic acid
molecules are RNA, including mRNA.
[0082] Definitions of common terms in cell biology and molecular
biology can be found in The Encyclopedia of Molecular Biology,
published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);
Benjamin Lewin, Genes X, published by Jones & Bartlett
Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8) and Current Protocols in Protein Sciences 2009,
Wiley Intersciences, Coligan et al., eds.
[0083] Unless otherwise stated, the present invention was performed
using standard procedures, as described, for example in Sambrook et
al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001);
Davis et al., Basic Methods in Molecular Biology, Elsevier Science
Publishing, Inc., New York, USA (1995); or Methods in Enzymology:
Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A.
R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); and
Current Protocols in Protein Science (CPPS) (John E. Coligan, et.
al., ed., John Wiley and Sons, Inc.), which are all incorporated by
reference herein in their entireties.
[0084] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While specific embodiments of, and examples for,
the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize. For example, while method steps or functions are
presented in a given order, alternative embodiments may perform
functions in a different order, or functions may be performed
substantially concurrently. The teachings of the disclosure
provided herein can be applied to other procedures or methods as
appropriate. The various embodiments described herein can be
combined to provide further embodiments. Aspects of the disclosure
can be modified, if necessary, to employ the compositions,
functions and concepts of the above references and application to
provide yet further embodiments of the disclosure. Moreover, due to
biological functional equivalency considerations, some changes can
be made in protein structure without affecting the biological or
chemical action in kind or amount. These and other changes can be
made to the disclosure in light of the detailed description. All
such modifications are intended to be included within the scope of
the appended claims.
[0085] Specific elements of any of the foregoing embodiments can be
combined or substituted for elements in other embodiments.
Furthermore, while advantages associated with certain embodiments
of the disclosure have been described in the context of these
embodiments, other embodiments may also exhibit such advantages,
and not all embodiments need necessarily exhibit such advantages to
fall within the scope of the disclosure.
[0086] The following examples are set forth as being representative
of the present disclosure. These examples are not to be construed
as limiting the scope of the present disclosure as these and other
equivalent embodiments will be apparent in view of the present
disclosure, figures and accompanying claims.
Example I
Linker Design
[0087] A test amyloid chimeric protein library was constructed to
test various linker designs between an amyloid domain (CsgA) and a
functional peptide domain (FLAG). The CsgA protein is secreted by
the bacterium Escherichia coli and the protein then self-assembles
into highly robust functional amyloid nanofibers with a diameter of
.about.4-7 nm. These amyloid fibers are known as `curli` and exist
as extended tangled networks encapsulating the cells. The FLAG
domain is an octapeptide polypeptide tag used for affinity
chromatography and epitope-tagged protein detection.
[0088] Six different domains were designed as linkers connecting
the CsgA and FLAG domains as set forth in Table 1 below.
TABLE-US-00001 Linker Plasmid Name Description Linker Full Sequence
pBbE1a-CsgA-F12- Flexible [GGGS]x3 F12 GGGSGGGSGGGS (SEQ ID NO: 5)
FLAG pBbE1a-CsgA-F24- Flexible [GGGS]x6 F24
GGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 6) FLAG pBbE1a-CsgA-F48-
Flexible [GGGS]x12 F48 GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS FLAG
(SEQ ID NO: 7) pBbE1a-CsgA- PolyProline [P]x12 P12 PPPPPPPPPPPP
(SEQ ID NO: 8) Pro12-FLAG pBbE1a-CsgA- PolyProline [P]x24 P24
PPPPPPPPPPPPPPPPPPPPPPPP (SEQ ID NO: 9) Pro24-FLAG pBbE1a-CsgA-EK3-
alpha-Helical EK3 EAAAKEAAAKEAAAK (SEQ ID NO: 10) FLAG [EAAAK]x3
pBbE1a-CsgA-EK9- alpha-Helical EK9
EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK FLAG [EAAAK]x9 (SEQ ID NO: 11)
indicates data missing or illegible when filed
[0089] According to one aspect, a linker may include from between 7
and 50 amino acids. According to one aspect, a linker may include
from between 8 and 50 amino acids. According to one aspect, a
linker may include from between 9 and 50 amino acids. According to
one aspect, a linker may include from between 10 and 50 amino
acids. According to one aspect, a linker may include from between
11 and 50 amino acids. According to one aspect, a linker may
include from between 12 and 50 amino acids. According to one
aspect, a linker may include from between 15 and 50 amino acids.
According to one aspect, a linker may include from between 20 and
50 amino acids. According to one aspect, a linker may include from
between 24 and 50 amino acids. According to one aspect, a linker
may include from between 45 and 50 amino acids.
[0090] According to one aspect, a linker may be flexible or rigid.
A linker may include one or more or a plurality of repeating amino
acid subunits.
[0091] According to one aspect, a linker may be hydrophobic or
hydrophilic.
[0092] According to one aspect, a linker may include 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 or greater amino acid subunits.
According to one aspect, a linker may include 2 repeating subunits
or more, 3 repeating subunits or more, 4 repeating subunits or
more, 5 repeating subunits or more, 6 repeating subunits or more, 7
repeating subunits or more, 8 repeating subunits or more, 9
repeating subunits or more, 10 repeating subunits or more, 11
repeating subunits or more, 12 repeating subunits or more, 13
repeating subunits or more, 14 repeating subunits or more, or 15
repeating subunits or more. According to one aspect, a plurality of
amino acid subunits results in a flexible linker. A flexible linker
may include a linker with a sequence that lacks inherent secondary
or tertiary structure in solution. According to one aspect, a
plurality of amino acid subunits results in a rigid linker. A rigid
linker may include a linker with a sequence that has a secondary or
tertiary structure that allows it to maintain a defined
conformation in solution.
[0093] An exemplary amino acid subunit is GGGS (SEQ ID NO:4).
According to one aspect, a linker has the structure [GGGS].sub.n
where n is an integer from 1 to 20 (SEQ ID NO:12). Exemplary values
for n include 3, 6, and 12. According to one aspect, a linker
having a plurality of GGGS (SEQ ID NO:4) subunits is a linker that
is flexible in whole or in part.
[0094] An exemplary amino acid subunit is P. According to one
aspect, a linker has the structure [P].sub.n where n is an integer
from 1 to 30 (SEQ ID NO:13). According to one aspect, linkers with
repetitive Ps include extended type II trans helices. Exemplary
values for n include 12 and 24. According to one aspect, a linker
having a plurality of P subunits is a linker that is rigid in whole
or in part.
[0095] An exemplary amino acid subunit is an alpha-helix motif such
as EAAAK (SEQ ID NO:14). According to one aspect, a linker has the
structure [EAAAK].sub.n where n is an integer from 1 to 15 (SEQ ID
NO:15). Exemplary values for n include 3 and 9. According to one
aspect, a linker having a plurality of EAAAK (SEQ ID NO:14)
subunits is a linker that is rigid in whole or in part and includes
a hydrophilic portion and a hydrophobic portion.
[0096] Exemplary amino acid residues include 12 or more amino
acids, 24 or more amino acids or 48 or more amino acids. According
to one aspect, the amino acids are flexible amino acids. According
to one aspect, the amino acids are one or more of glycine, serine,
alanine or leucine. According to one aspect, a corresponding
increase in extracellular curli fibers results from increased
linker length. According to one aspect, a corresponding increase in
functional domain accessibility results from increased linker
length.
[0097] Exemplary linkers include:
TABLE-US-00002 (SEQ ID NO: 16) GGGSGGGSGGGS, (SEQ ID NO: 16)
GGGSGGGSGGGSGGGSGGGSGGGS, (SEQ ID NO: 16)
GGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 17)
PPPPPPPPPPPP, (SEQ ID NO: 17) PPPPPPPPPPPPPPPPPPPPPPPP, (SEQ ID NO:
18) EAAAKEAAAKEAAAK, or (SEQ ID NO: 18)
EAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAK.
Example II
Analysis of Amyloid Nanofiber Production
[0098] CsgA-linker-FLAG chimeras with different intervening linkers
were tested for optimal self-assembly into curli nanofibers. The
self-assembly of the protein library with different linkers into
curli nanofibers was detected using Congo Red (CR), a standard
stain used for the colorimetric detection of amyloids. Only E. coli
cells expressing fusion proteins that are successfully secreted and
are competent for self-assembly into amyloid nanofibers will stain
red. As shown in FIG. 1, all the flexible linkers (F12, F24, and
F48) show some intermediate levels of CR staining, with the longest
linker showing the greatest amount. For the rigid linkers, the
longer polyproline linker showed no CR staining and the highest
amount of CR staining was obtained for the EK3 linker. A trend is
seen for the polyproline and EAAAK (SEQ ID NO:14) linkers with the
longer rigid linkers resulting in less staining. It is to be
understood that this is a representative embodiment only and that a
polyproline linker having 24 prolines may be a useful linker for a
given functional polypeptide.
Example III
Determination of Peptide Domain Functionality
[0099] To determine if the FLAG epitope tag is accessible and can
properly perform its prescribed function, which is binding to an
anti-FLAG antibody, immunoblot analysis of whole-cell culture
samples was performed. The CsgA-linker-FLAG structure with a
flexible linker demonstrated a monotonic increase in fluorescence
as a function of the linker length. The CsgA-linker-FLAG structure
with a polyproline rigid linker lacked fluorescence. Without
wishing to be bound by scientific theory, these hydrophobic linkers
may impede proper functioning of the peptide tag. The
CsgA-linker-FLAG structure with an alpha-helical EAAAK (SEQ ID
NO:14) linker showed that the EK3 linker construct, which had more
CR staining than the EK9 construct (see FIG. 1), had lower
fluorescence. In contrast, the lower CR-binding EK9 linker
construct had a high fluorescent signal, indicating greater
accessibility of the FLAG epitope tag. See FIG. 2.
Example IV
Method of Optimizing Linker Design
[0100] Aspects of the present disclosure are directed to methods of
optimizing linker design for linking CsgA and a functional
polypeptide. According to this aspect, a functional polypeptide is
selected for linkage with CsgA. A linker design is then selected to
form the CsgA-linker-functional polypeptide structure. A bacteria
is then modified to include a nucleic acid sequence encoding the
CsgA-linker-functional polypeptide structure. The modified bacteria
may then be caused to proliferate into a population of bacteria and
is assayed for the expression of the CsgA-linker-functional
polypeptide structure. The modified bacteria may then be caused to
proliferate into a population of bacteria and is assayed for the
formation of a biofilm of the CsgA-linker-functional polypeptide
structure. The biofilm may be assayed for the functional
characteristics of the functional polypeptide. Accordingly,
exemplary linkers described herein allow the self-assembly and
functional domains of the CsgA-linker-functional polypeptide
structure to operate or function without interfering with each
other. An exemplary method of determining the effect of a linker on
biofilm formation and functional characteristics of the functional
polypeptide is depicted in FIG. 3. Unoptimized linker domains may
hinder the performance of the biofilm-based material by occluding
binding sites for the functional domains, preventing proper folding
of the CsgA or functional polypeptide domains, hindering assembly
of amyloid fibers or hindering secretion. Optimized linkers
minimize these issues and improve the desired function of the
biofilm. Optimized linker domains facilitate the fabrication of
biofilm based materials with superior mechanical and biophysical
stability under a wide range of conditions (extreme temperatures,
extreme pH, in the presence of unfavorable solvents, under exposure
to the elements in outdoor applications, etc.). Optimized linker
domains enable each functional peptide domain to operate
independently, effectively increasing the number of functional
peptide domains in the material that are contributing to a desired
behavior on the micro- or macro-scale. For example, if the desired
function of the biofilm-based material is metal binding, the
material may have inhibited stability or metal binding affinity
because of molecular crowding or undesirable molecular interactions
that result from inappropriate linker domains Optimized linker
domains reduce undesirable molecular interactions from occurring,
ultimately enhancing the performance of the material.
Example V
Gut Epithelial Binding
[0101] Aspects of the present disclosure are directed to methods of
using engineered bacteria to create a biofilm having a heterologous
domain that binds to specific tissues or cells whether healthy or
diseased. According to one aspect, the bacteria is a non-pathogenic
bacteria. Exemplary non-pathogenic bacteria include Nissle strain
1917 (EcN), MG1655, K12-derived strains, PHL628, LSR10, LSR6 and
the like. Wild-type EcN, which is marketed as a probiotic under the
trade name Mutaflor, can be delivered orally, survive transit
through the upper GI tract, then transiently colonize the ileum and
colon for several days after initial administration. During this
colonization process, EcN produces curli fibers.
[0102] Exemplary binding domains include those identified in Table
2 below.
TABLE-US-00003 Name Sequence Origin Function CP15 VHLGYAT (SEQ ID
NO: 19) Phage Display Binds Colon Carcinomas P8 LETTCASLCYPS (SEQ
ID NO: 20) Phage Display Binds M cells A1 VRPMPLQ (SEQ ID NO: 21)
Phage Display Binds Colon Carcinomas T18 LTHPQDSPPASA (SEQ ID NO:
22) Phage Display Binds Injured Epithelium TFF1
EAQTETCTVAPRERQNCGFPGVTPSQCANKGCCFDDTV Trefoil Factor Anti-
RGVPWCFYPNTIDVPPEEECEF (SEQ ID NO: 39) inflammatory Binds mucosa
TFF2 EKPAACRCSRQDPKNRVNCGFPGITSDQCFTSGCCFDSQ Trefoil Factor Anti-
VPGVPWCFKPLPAQESEECVMQVSARKNCGYPGISPED inflammatory
CAARNCCFSDTIPEVPWCFFPMSVEDCHY Binds mucosa (SEQ ID NO: 23) TFF3
EEYVGLSANQCAVPAKDRVDCGYPHVTPKECNNRGCC Trefoil factor Anti-
FDSRIPGVPWCFKPLQEAECTF (SEQ ID NO: 24) inflammatory Binds mucosa
Lunasin SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDD Soybean 2S Anti- DD
DDDD (SEQ ID NO: 32) albumin inflammatory MAM
MMMPANYSVIAENEMTYVNGGANFIDAIGAVT Faecalibacterium Anti-
APIWTLDNVKTFNTNIVTLVGNTFLQSTINRTIVL prausnitza inflammatory
FSGNTTWKEVGNIGKNLFGTNVKGNPIEKNNFGDYAMN
ALGIAAAVYNLGVAPTKNTVKETEVKFTV (SEQ ID NO: 33)
[0103] We have identified specific combinations of linker sequences
and culture conditions that lead to optimal curli fiber formation
for each of the different fusion domains is provided in Table 3
below.
TABLE-US-00004 Optimal linker Optimal CsgA-peptide domain [IPTG]
Temperature Duration construct (SEQ ID NO: 40) (mM) (C.) (h)
CsgA-TFF1 48 AA (GGGS).sub.n 0.3 37 24 CsgA-TFF2 24 AA (GGGS).sub.n
0.3 37 24 CsgA-TFF3 24 AA (GGGS).sub.n 0.3 37 24 CsgA-lunasin 36 AA
(GGGS).sub.n 0.3 37 24 CsgA-MAM 36 AA (GGGS).sub.n 0.3 37 24
CsgA-T18 48 AA (GGGS).sub.n 0.3 25 48 CsgA-CP15 48 AA (GGGS).sub.n
0.3 25 48 CsgA-P8 48 AA (GGGS).sub.n 0.3 25 48
[0104] Functional polypeptides include a polypeptide or protein
sequence that displays binding affinity with the epithelial
surfaces of the gastrointestinal (GI) tract. Exemplary polypeptide
or protein sequences are known to those of skill in the art or can
be determined from phage display on biological tissues, from
sequences that exist in naturally occurring organisms or from
sequences that have been engineered in some other way to bind to
specific surfaces. For example, exemplary polypeptide or protein
sequences having affinity with tissues or cells associate with the
GI tract can be determined from a microfluidic system that mimics
the structure of a gut epithelium. Such systems may be referred to
as a "Gut on a CHIP." Such systems incorporate flow and cyclic
strain motions to mimic peristalsis. Engineered bacteria strains as
described herein having gut binding functional groups may be
introduced into such a system and the micro-scale localization and
residence time in the system may be monitored to determine binding
of the cells through expression of the CsgA-linker-functional
polypeptide to the system. Such a system is described in Lab Chip,
2012, 12, 2165-2174, hereby incorporated by reference in its
entirety.
[0105] Exemplary polypeptide or protein sequences having affinity
with tissues or cells associate with the GI tract can be determined
from monitoring or determining spatiotemporal distribution in mouse
models. According to this aspect, engineered bacteria strains, such
as engineered Nissle strains, are administered orally, such as a
single dose, to the healthy mouse gut followed by a period of
normal feeding. Residence time of the engineered strains will be
measured by CFU counting from fecal samples collected daily.
Spatial localization within the gut will be monitored by harvesting
the gut tissue, sectioning, and tracking the presence of engineered
strains by CFU counts from homogenized tissues and immune staining
of histological tissue slices.
[0106] Several linkers were tested in a library of polypeptide
domains that are known to bind to gut epithelia in a localized
manner Known as trefoil factors (TFFs), this polypeptide family
consists of 3 identified peptides (TFF1, TFF2, and TFF3), that are
secreted by the gastrointestinal mucosa and differentially bind to
different areas of the gastrointestinal tract. All trefoil factors
have in common a trefoil domain, which consists of three conserved
disulfide bonds. The sequence for each TFF construct engineered
into a CsgA-linker is shown in Table 2 above.
[0107] TFF1 contains a single trefoil domain of 60 amino acids.
TFF2 contains two homologous trefoil domains, resulting in a total
of 6 disulfide bonds, and is over 100 amino acids in length.
Various constructs were made containing CsgA fused to the TFF2 via
the flexible linkers identified in the preceding experiments. For
the single trefoil domain construct (TFF1), longer flexible linker
domains resulted in a marked improvement in expression levels as
measured by a quantitative Congo Red binding assay (FIG. 4).
[0108] TFF1 showed nearly a 5-fold increase when the flexible
linker length was increased from 24 to 48 residues. In contrast,
TFF2 showed no improvement as a function of linker length, which
may be due to the complexity of the protein (6 disulfides) or
larger length (>100 amino acids).
[0109] Various short peptides (7-12 amino acids) were tested, using
the F48 linker as shown in Table 4.
TABLE-US-00005 TABLE 3 BIND Variants for Small Peptide Gut
Epithelial Tissues Peptide domain Plasmid Name Sequence Gut
Localization Ref. pBbEla-CsgA-F48- LTHPQDSPPASA Injured epithelial
Costantini TW, et T18 (SEQ ID NO: 25) cells al..sup.1
pBbE1a-CsgA-F48- VHLGYAT Colon cancer Zhang Y, et al..sup.2 CP15
(SEQ ID NO: 26) pBbE1a-CsgA-F48- LETTCASLCYPS M cells, FAE Higgins
L et al..sup.3 P8 (SEQ ID NO: 27) pBbE1a-CsgA-F48- VRPMPLQ Colon
cancer Hsiung PL et al..sup.4 A1 (SEQ ID NO: 28)
[0110] These peptides have been identified through phage-display
techniques for binding to the intestinal mucosa. As shown in FIG.
5, all of these short peptide constructs demonstrated export and
curli-self-assembly levels comparable to that of the wild type
CsgA.
[0111] To test if these various gut-binding domains (TFFs and the
short peptides) retained their tissue homing functions when
displayed on curli nanofibers, standard in vitro assays for
bacterial binding to Caco-2 cell monolayers was performed. Caco-2
cell lines are phenotypically similar to the enteric columnar
epithelial cells that line the human small intestine. These cell
lines are widely used in research laboratories as model in vitro
systems. Various CsgA constructs, all containing the F48 linker
domain, were expressed in the E. coli LSR10 strain. This strain of
E. coli is a laboratory K-12 strain which produces no other
extracellular organelles (cellulose, flagella, pili, etc.), thus
allowing for clear discernment of function. FIG. 6 shows adhesion
of LSR10 cells expressing CsgA constructs with gut-binding domains
to Caco-2 cells. Various CsgA constructs, all containing the F48
linker domain, were expressed in the E. coli PHL628 strain. FIG. 7
shows adhesion of PHL628 cells expressing CsgA constructs with
gut-binding domains to Caco-2 cells.
[0112] The expression and binding of the constructs were further
tested in a probiotic strain of E. coli, Nissle 1917, which
expresses curli nanofibers. The Nissle strain was isolated during
WWI from the fecal samples of a soldier who was resistant to
infectious enteropathogenic diarrhea. Further studies have shown
the Nissle strain to be a profound probiotic, with the ability to
protect against gastroinvasive bacteria and to ameliorate other
gastrointestinal disorders such as ulcerative colitis, irritable
bowel syndrome, and Crohn's disease. Accordingly, aspects of the
present disclosure are directed to attaching Nissle E. coli to gut
tissue.
[0113] The endogenous CsgA gene was removed from the Nissle strain
using the Lambda Red genomic deletion technique, resulting in an
engineered strain of Nissle (Nissle mutant, PBP17) with a precise
csgA deletion. FIG. 8A-8D shows construction of a CsgA deletion
mutant of Nissle 1917. FIG. 8A shows a lambda red recombination
strategy for the deletion of the csgA gene in Nissle. FIG. 8B shows
PCR validation of the chloramphenicol cassette insertion at the
csgA locus identifies two positive clones (black arrows). FIG. 8C
shows two clones verified for the presence of the 265, 158, and 113
bp amplicons. FIG. 8D shows sequencing verification of the regions
flanking the csgA gene indicates successful CAT cassette
integration (highlighted in yellow). To test the adhesion
functionality of the various constructs expressed in PBP17, an in
vitro binding study to Caco-2 cells was performed as described
above. Constructs with the F48 linker and containing trefoil
domains (TFF1, TFF2, and TFF3) were expressed in a Nissle
.DELTA.csgA mutant (PBP17) and tested for in vitro binding to
Caco-2 monolayers. Bound cells were recovered and quantified by CFU
analysis and the data is presented in FIG. 9 as the % of total
inoculated bacterial cells that remained adhered to the Caco-2
monolayer.
[0114] Exemplary structures include
TABLE-US-00006 (SEQ ID NO: 41) CsgA-(GGGS).sub.n-TTF1, wherein n is
3, 6 or 9. (SEQ ID NO: 41) CsgA-(GGGS).sub.n-TTF2, wherein n is 3,
6 or 9. (SEQ ID NO: 41) CsgA-(GGGS).sub.n-TTF3, wherein n is 3, 6
or 9. (SEQ ID NO: 41) CsgA-(GGGS).sub.n-T18, wherein n is 3, 6 or
9. (SEQ ID NO: 41) CsgA-(GGGS).sub.n-CP15, wherein n is 3, 6 or 9.
(SEQ ID NO: 41) CsgA-(GGGS).sub.n-P8, wherein n is 3, 6 or 9.
Example VI
Treating Chronic Inflammatory Diseases of the Gut
[0115] Aspects of the present disclosure are directed to methods of
treating chronic inflammatory diseases of the gut, such as
inflammatory bowel disease and Crohn's disease by administering to
an individual in need thereof a certain engineered bacterial strain
or strains as described herein. According to one aspect, the
bacterial strain expresses a CsgA-linker-anti-inflammatory
polypeptide construct.
[0116] The bacterial strain expresses the
CsgA-linker-anti-inflammatory polypeptide construct and the
anti-inflammatory polypeptide treats the chronic inflammatory
disease of the gut.
[0117] According to one aspect, the bacterial strain expresses a
CsgA-linker-first member of a binding pair polypeptide construct.
The second member of the binding pair attached to an
anti-inflammatory compound is then administered. The first member
of the binding pair and the second member of the binding pair bind
thereby localizing the anti-inflammatory compound, for example, to
the gut, for the treatment of chronic inflammatory diseases of the
gastrointestinal tract.
[0118] According to one aspect, the bacterial strain is a
non-pathogenic bacterial strain, such as the Nissle strain or any
other non-pathogenic strain known to those of skill in the art such
as MG1655, K12-derived strains, and the like.
[0119] The anti-inflammatory compound can be a protein sequence
capable of abrogating inflammatory processes in the gut. Exemplary
protein sequences include the trefoil factor family of peptides
(TFFs) because they are endogenous signaling molecules in the
mammalian gut, have demonstrated efficacy in treating IBD and
Crohn's in the clinic, and are under development in their soluble
form as biologics for these indications.
[0120] According to one aspect, the anti-inflammatory compound,
protein or polypeptide may exert its effects while still attached
to the engineered curli fibers. According to one aspect, the
anti-inflammatory compound, protein or polypeptide may exert its
effects as a soluble protein or compound after cleavage from the
curli fibers by a protease. Several proteases in the MMP family
(MMP2, MMP9 and the like) are upregulated during inflammation and
may be used to cleave the anti-inflammatory compound, protein or
polypeptide from the linker domain.
[0121] According to one aspect, methods are provided to deliver
bioactive proteins and peptides locally to the inflamed gut.
According to one aspect, methods are provided to deliver bioactive
proteins and peptides locally to the inflamed gut in response to
specific inflammatory cues.
[0122] Anti-inflammatory compound, protein or polypeptide may be
identified by the exemplary methods described below for TTF
domains. Caco-2 intestinal epithelial cells grown in 2D culture are
treated with purified curli fibers displaying TFF domains.
Biomarkers indicative of TFF bioactivity are monitored such as cell
migration speed as TFFs are known to promote cell migration. The
migration speeds for assembled curli fibers composed of either
wt-CsgA (neg. ctrl), CsgA-TFF, or soluble TFF (positive ctrl) are
compared. Migration speeds are measured using a wound healing assay
on confluent monolayers. If TFF is active, it will increase
migration speeds, comparable to the soluble TFFs.
[0123] According to another method, levels of COX-2 expression in
the three cell populations are compared by Western blot. Expression
above the negative control confirms the bioactivity of the
curli-bound TFFs.
[0124] According to another method, a Gut-on-a-Chip system is used
to measure the bioactivity of the curli-bound TFFs using a more
physiologically-relevant model system. Chronic inflammation is
simulated by adding inflammatory agents (e.g. LPS) to a chip
containing an epithelial layer, endothelial layer, and circulating
immune cells. See Kim et al., LabChip 12, 2165 (2012) and Kim et
al., Integrative biology: quantitative Biosciences from nano to
Macro 5, 1130 (2013). The expression of key inflammatory markers
(NF-.kappa.B, IL-1.beta., IL-8, COX-2, EGFR activation, etc.) is
then monitored in response to treatment with curli-bound TFFs.
[0125] According to another method, established mouse models of
intestinal inflammation (DSS, IL-10 knockout, etc.) are used to
test the anti-inflammatory effects of the curli-bound TFFs.
Abrogated inflammatory responses are measured qualitatively by
histology and quantitatively with qPCR of key inflammatory
cytokines.
Example VII
Treating Cancers of the Gut
[0126] Aspects of the present disclosure are directed to methods of
treating cancer such as cancer of the GI tract by administering to
an individual in need thereof a certain engineered bacterial strain
or strains as described herein. According to one aspect, the
bacterial strain expresses a CsgA-linker-cancer treating
polypeptide construct. Certain cancer treating polypeptides include
those having known activity against cancers including growth
inhibiting factors such as bevacizumab, cetuximab, panitumumab and
the like.
[0127] The bacterial strain expresses the CsgA-linker-cancer
treating polypeptide construct and the cancer treating polypeptide
treats the cancer tissue or cancer cells. According to one aspect,
the bacterial strain expresses a CsgA-linker-first member of a
binding pair polypeptide construct. The bacterial strain expresses
the CsgA-linker-first member of a binding pair polypeptide
construct. The second member of the binding pair attached to a
cancer treating compound is then administered. The first member of
the binding pair and the second member of the binding pair bind
thereby localizing the cancer treating compound, for example, to
the gut, for the treatment of cancer, such as cancers of the
gastrointestinal tract.
[0128] Useful bacterial strains include non-pathogenic E. coli,
such as the E. coli Nissle 1917 (EcN), MG1655, K12-derived strains
and the like.
[0129] According to one aspect, the cancer treating protein or
polypeptide may exert its effects while still attached to the
engineered curli fibers. According to one aspect, the cancer
treating protein or polypeptide domain may exert its effects as a
soluble protein after cleavage from the curli fibers by a protease.
Several proteases in the MMP family are upregulated during
inflammation and may be used to cleave the cancer treating
polypeptide from the linker domain.
[0130] Methods described above can be used to confirm both in vitro
and in vivo activity of this embodiment of the disclosure.
Example VIII
Diagnostic Methods
[0131] Aspects of the present disclosure are directed to methods of
delivering a diagnostic agent, such as a marker, to a site within a
mammal by administering to the mammal a certain engineered
bacterial strain or strains as described herein. According to one
aspect, the bacterial strain expresses a CsgA-linker-diagnostic
polypeptide construct.
[0132] The bacterial strain expresses the CsgA-linker-diagnostic
polypeptide construct and the diagnostic polypeptide is detected.
According to one aspect, the bacterial strain expresses a
CsgA-linker-first member of a binding pair polypeptide construct.
The bacterial strains express the CsgA-linker-first member of a
binding pair polypeptide construct. The second member of the
binding pair attached to a diagnostic compound is then
administered. The diagnostic compound may include an imaging agent
or dye. The first member of the binding pair and the second member
of the binding pair bind thereby localizing the diagnostic compound
to the location of interest, such as the gut.
[0133] Useful bacterial strains include non-pathogenic E. coli,
such as the Nissle strain, MG1655, K12-derived strains and the
like.
[0134] According to one aspect, the diagnostic polypeptide or
diagnostic compound may be still attached to the engineered curli
fibers. According to one aspect, the diagnostic polypeptide or the
diagnostic compound may be cleaved from the curli fibers by a
protease. Several proteases in the MMP family are upregulated
during inflammation and may be used to cleave the diagnostic
polypeptide or diagnostic compound from the linker domain.
[0135] According to one aspect, the diagnostic polypeptide or
diagnostic compound may be released from the curli fibers using a
protease-cleavable linker. According to this aspect, a recombinant
construct of CsgA-linker-diagnostic polypeptide is made where the
linker domain includes an amino acid sequence that is recognized
and cleaved by a protease enzyme. Such amino acid sequences and
associated protease enzymes are known to those of skill in the art
and include MMPs, CD2830 and the like. The linker may be selected
such that it is susceptible to cleavage by enzymes that are
produced locally at sites of inflammation (MMP2, MMP9, etc.).
Methods described above can be used to confirm both in vitro and in
vivo activity of this embodiment of the disclosure.
Example IX
Treatment of Gut Borne Pathogens
[0136] Aspects of the present disclosure are directed to methods of
treating gut borne pathogens by administering to an individual in
need thereof a certain engineered bacterial strain or strains as
described herein. According to one aspect, the bacterial strain
expresses a CsgA-linker-antimicrobial polypeptide construct.
Certain antimicrobial polypeptides or proteins include any
polypeptide or protein sequence having antimicrobial activity.
Antimicrobial peptides for use in a therapeutic context are known
to those of skill in the art. See Cotter, P. D., Ross, R. P. &
Hill, C. Bacteriocins--a viable alternative to antibiotics? Nat Rev
Micro 11, 95-105 (2012); Hing, T. C. et al. The antimicrobial
peptide cathelicidin modulates Clostridium difficile-associated
colitis and toxin A-mediated enteritis in mice. Gut 62, 1295-1305
(2013); Nuding, S., Frasch, T., Schaller, M., Stange, E. F. &
Zabel, L. T. Synergistic Effects of Antimicrobial Peptides and
Antibiotics against Clostridium difficile. Antimicrobial Agents and
Chemotherapy 58, 5719-5725 (2014); Rea, M. C. et al. Thuricin CD, a
posttranslationally modified bacteriocin with a narrow spectrum of
activity against Clostridium difficile. Proc Natl Acad Sci USA 107,
9352-9357 (2010); Petrof, E. Probiotics and Gastrointestinal
Disease: Clinical Evidence and Basic Science. AIAAMC 8, 260-269
(2009). Kang, J. K. et al. The Insect Peptide Coprisin Prevents
Clostridium difficile-Mediated Acute Inflammation and Mucosal
Damage through Selective Antimicrobial Activity. Antimicrobial
Agents and Chemotherapy 55, 4850-4857 (2011); and Ostaff, M. J.,
Stange, E. F. & Wehkamp, J. Antimicrobial peptides and gut
microbiota in homeostasis and pathology. EMBO Mol Med 5, 1465-1483
(2013).
[0137] According to one aspect, gut borne pathogens include
Clostridium difficile, Salmonella typhimurium, Enteropathogenic E.
coli, Helicobacter pylori and the like.
[0138] The bacterial strain expresses the CsgA-linker-antimicrobial
polypeptide construct and the antimicrobial polypeptide treats the
gut borne pathogens in a manner to reduce or eliminate the gut
borne pathogens. According to one aspect, the bacterial strain
expresses a CsgA-linker-tissue or cell binding polypeptide
construct. The bacterial strain expresses the CsgA-linker-tissue or
cell binding polypeptide construct and the bacterial strain treats
the gut borne pathogens in a manner to reduce or eliminate the gut
borne pathogens. According to one aspect, the bacterial strain
expresses a CsgA-linker-first member of a binding pair polypeptide
construct. The bacterial strains express the CsgA-linker-first
member of a binding pair polypeptide construct. The second member
of the binding pair attached to an antimicrobial compound is then
administered. The first member of the binding pair and the second
member of the binding pair bind thereby localizing the
antimicrobial compound to the gut for the treatment of the gut
borne pathogens in a manner to reduce or eliminate the gut borne
pathogens.
[0139] Useful bacterial strains include non-pathogenic E. coli,
such as the E. coli Nissle 1917 (EcN), MG1655, K12-derived strains
and the like.
[0140] According to one aspect, the antimicrobial protein or
polypeptide may exert its effects while still attached to the
engineered curli fibers. According to one aspect, the antimicrobial
protein or polypeptide may exert its effects as a soluble protein
after cleavage from the curli fibers by a protease. Several
proteases in the MMP family are upregulated during inflammation and
may be used to cleave the bioactive domain from the linker
domain.
[0141] According to one aspect, the antimicrobial proteins or
peptides may be released from the curli fibers using a
protease-cleavable linker. According to this aspect, a recombinant
construct of CsgA-linker-antimicrobial is made where the linker
domain includes an amino acid sequence that is recognized and
cleaved by a protease enzyme. Such amino acid sequences and
associated protease enzymes are known to those of skill in the art.
The linker may be selected such that it is susceptible to cleavage
by enzymes that are produced locally at sites of inflammation
(MMP2, MMP9, etc.). According to one aspect, antimicrobial proteins
are fused to the curli fibers via a linker that is susceptible to
cleavage by the proteolytic virulence factor CD2830. According to
one aspect, antimicrobial proteins are released inside the gut only
in the presence of C. difficile virulence factors. The
antimicrobial protein is delivered locally to kill the invading
pathogen.
[0142] Methods described above can be used to confirm both in vitro
and in vivo activity of this embodiment of the disclosure.
[0143] There are several examples of antimicrobial proteins with
demonstrated activity against C. difficile, including thurcin CD,
lantibiotics like nisin and actagardine, cathelicidins and LL-37.
One exemplary antimicrobial protein is coprisin, a peptide that was
originally isolated from Copris tripartitus (a Korean dung beetle)
that has recently shown promise as a treatment for C. difficile
infections. Coprisin has a high potency against C. difficile (MIC
of 1.5 .mu.g/mL compared to 3.0 .mu.g/mL for vancomycin) and a lack
of activity against common gut commensals like Lactobacillus and
Bifidobacterium. The full coprisin peptide is 43 amino acids, but a
9 amino acid truncated analog (LLCIALRKK)(SEQ ID NO:29) exhibits
higher antibiotic activity. The coprisin-derived sequence is fused
to CsgA through a linker that is susceptible to cleavage in the
presence of a protease virulence factor secreted by C. difficile
during infection. See Hensbergen, P. J. et al. A novel secreted
metalloprotease (CD2830) from Clostridium difficile cleaves
specific proline sequences in LPXTG (SEQ ID NO:30) cell surface
proteins. Molecular & Cellular Proteomics 13, 1231-1244 (2014).
CD2830 is a metalloprotease that is actively secreted by C.
difficile into the extracellular space and is thought to play a
role in pathogen motility by cleaving adhesions that bind to the
epithelial cell surface. Importantly, CD2830 is known to exhibit
specificity for cleavage of proline-rich sequences, especially
Pro-Pro and sortase-like LPXTG (SEQ ID NO:30) sequences.
[0144] A panel of CsgA-linker-coprisin variants is constructed
using a combination of direct synthesis of peptide inserts (via
Integrated DNA Technologies) and overlap extension PCR. The gene
panel will vary in the length and sequence of the linker region as
shown in FIG. 10 in order to identify an optimal sequence for
export and cleavage. Proline-rich regions were selected based on
the fastest cleavage rates when subjected to CD2830, as reported by
Hensbergen, et al. FIG. 10 is a diagram of CsgA-linker-AMP
constructs for synthesis and screening. Sec is a periplasmic
secretion tag that is cleaved after transport. N22 is the outer
membrane secretion tag. CsgA is the amyloidogenic region of the
protein. Linkers have the general sequence (GGS).XXXX, with n=3, 4,
or 5 and XXXX=PPP, PPIP, or PPVP (SEQ ID NO:31). The total panel
size is 9 members, with fused domains ranging from 22-28 amino
acids.
[0145] These genes are incorporated into the appropriate plasmids
using previously reported protocols. The plasmids harbor ampicillin
resistance genes for antibiotic selection and the genes encoding
for the CsgA fusion proteins are placed under the control of an
IPTG inducible promoter.
[0146] Genetic constructs are made and introduced into EcN and the
ability of EcN to synthesize, secrete, and assemble curli fibers
displaying the linker-AMP domains is tested. Plasmids encoding for
the CsgA-linker-AMP constructs are transformed into a mutant of EcN
in which the genomic copy of the csgA gene has been deleted and
replaced with an antibiotic selection marker (EcN-.DELTA.csgA).
This mutant is unable to produce curli fibers of its own, so any
curli-related signals obtained after plasmid transformation arise
from the engineered curli constructs. Congo Red staining is used to
rapidly screen for curli production in engineered E. coli variants.
In addition, curli production is assayed using a whole-cell ELISA
assay wherein induced EcN-.DELTA.csgA transformants are filtered
onto a membrane and probed directly with anti-CsgA antibodies.
Transformants are characterized using SEM to confirm that the
morphology of the modified curli fibers is qualitatively similar to
the wild-type fibers. In order to confirm that the linker-AMP
domains remain intact and have not been degraded during the
secretion and assembly process, an established purification
protocol is used to obtain pure curli fibers, which are
disassembled and subjected to MALDI-mass spectrometry analysis.
Comparison between growth rates of the various tranformants and
EcN-.DELTA.csgA in minimal media is used as a proxy for
fitness.
[0147] According to one aspect, the bioactive peptide is cleaved
and released from the curli fibers in response to a protease.
Suitable proteases can be identified using the following method
which is exemplified by protease CD2830. The CD2830 protease is
actively secreted by C. difficile, and is purported to act as a
virulence factor by cleaving host protein-binding adhesions
produced by the pathogen, thereby promoting motile phenotypes.
CD2830 cleaves proline-rich sequences. Accordingly, linker domains
include one or more prolines.
[0148] CD2830 is expressed recombinantly in E. coli and purified. A
96-well filter plate assay is used to subject biofilms to various
conditions and to monitor the capture or release of soluble
entities from the biofilm. A panel of engineered EcN variants are
grown and induced in suspension culture, then immobilized with
their associated curli fibers on the filter plate. The biofilms are
washed to remove all soluble or weakly bound biomolecules. The
biofilms are treated with the recombinant CD2830 at various
concentrations. Release of the AMPs is monitored at a range of time
points to determine the kinetic parameters of the cleavage from
assembled curl fibers. AMP release is monitored by LC-MS analysis
of the collected fractions after protease treatment. Similar
release studies is performed with a live C. difficile strain (ATCC
43255) by exposure of the filtered biofilms to the pathogen inside
an anaerobic chamber.
[0149] Antimicrobial activity of antimicrobial proteins, such as
coprisin, by two different assays of the modified curli fibers--one
that mimics a conventional minimum inhibitory concentration (MIC)
assay, and another that monitors the cytotoxicity of C. difficile
cells after culture in the presence of modified curli fibers. In
the first assay, EcN variants that are selected for their ability
to release active AMP in response to recombinant CD2830 are induced
in YESCA media to form modified curli fibers. The induced cell
cultures are grown to an OD.sub.600=1, then heat-treated to kill
the EcN cells before being transferred to an anaerobic chamber. C.
difficile cultures are prepared by growing them overnight under
anaerobic conditions to stationary phase. They are then be
incubated with the AMP-displaying curli fibers at various
concentrations for a range of times. Finally, viable C. difficile
cells are counted by plating serial dilutions on agar plates with
the appropriate growth medium and counting colony forming units
(CFUs). In the second assay, the ability of engineered EcN variants
to protect human epithelial cell layers from invasion by vegetative
C. difficile is tested. Using a published protocol, the colorectal
cancer-derived Caco-2 cell line (ATCC HTB37) is grown to confluency
in 96-well plates. See Wagner, R. D., Johnson, S. J. &
Cerniglia, C. E. In Vitro Model of Colonization Resistance by the
Enteric Microbiota: Effects of Antimicrobial Agents Used in
Food-Producing Animals. Antimicrobial Agents and Chemotherapy 52,
1230-1237 (2008).; Banerjee, P., Merkel, G. J. & Bhunia, A. K.
Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit
cytotoxic effects and adhesion of pathogenic Clostridium difficile
to Caco-2 cells. Gut Pathog 1, 8 (2009). The cell lines are then
incubated with the engineered EcN variants for 3 hours before being
washed with cell culture media to remove non-adhered cells. The
cell monolayers are exposed to vegetative C. difficile cells under
aerobic conditions and co-incubated for 3 hours. Cytotoxicity is
monitored using a LIVE/DEAD fluorescent staining assay coupled with
image-based detection of cell counts.
[0150] The altered CsgA genes are incorporated into the genome of
EcN. A mutant of EcN is generated wherein the csgA gene has been
replaced with an antibiotic selection marker by using a lambda Red
recombineering technique wherein a double stranded DNA insert
containing a desired selection marker flanked by homology domains
specific for the csgA locus is introduced into the bacterial cells
along with the lambda Red recombination factors. See Datsenko, K.
A. & Wanner, B. L. One-step inactivation of chromosomal genes
in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA
97, 6640-6645 (2000). The genome edited clones are selected by
plating on antibiotic selective plates and the presence of the
correct insertion is confirmed by PCR and sequencing. The resulting
strain, referred to as EcN-.DELTA.csgA, does not produce curli
fibers. A similar lambda Red recombineering strategy is used to
replace the csgA gene in wild-type EcN with genes encoding for
CsgA-linker-AMP variants. The genetic insertion cassettes contain
both the chimeric sequences and an antibiotic resistance marker for
the selection of successfully edited clones. The resistance gene is
flanked by flippase recognition target (FRT) domains that enable
self-resection if treated by mild heat. Such a method provides
"scarless" insertion of csgA variants.
[0151] Successfully edited clones are subjected to a range of
characterization protocols to ensure that they produce curli
fibers. Whole cell ELISA is used to confirm the presence of
assembled CsgA in the fibers. SEM is used to confirm that the gross
morphology of the engineered fibers resembles that of wild-type
curli fibers. MALDI-MS analysis of purified curli fibers is used to
confirm that the linker and AMP domains are not degraded during the
secretion and assembly process. Finally, growth rates in suspension
culture are monitored for wild-type EcN, EcN-.DELTA.csgA, and each
of the CsgA-linker-AMP constructs.
[0152] The viability of the EcN genomic mutants in the healthy
mouse gut is tested. The engineered EcN mutants survive and
transiently colonize the mouse gut without harming the host.
Genomically altered EcN mutants are fed to healthy mice at CFU
values of .about.108. The inoculation occurs once, and the mice are
fed a normal diet for 10 days. The residence time of the engineered
EcN variants in the mouse gut is monitored by collecting fecal
samples daily and counting viable colonies on Macconkey agar plates
with the appropriate antibiotic selection. Mice are observed and
their body weight measured daily in order to confirm that there are
no symptoms of bacterial infection. At the experimental endpoint,
the mice are sacrificed and their organs harvested in order to
determine the spatial distribution of EcN cells. The GI tract is
sectioned into upper, lower, and middle sections and homogenized
before counting CFUs by serial dilution on selective plates. A
similar protocol is applied to the liver and spleen to check for
invasive phenotypes.
[0153] The therapeutic effects of the engineered EcN variants on
gut inflammation following C. difficile infection are monitored
using the mouse model of antibiotic-induced C difficile-associated
disease (CDAD) developed by Kang, et al., which more closely
resembles human disease responses compared to other available
models. Mice in similar test groups are housed together and
pre-treated with water containing a cocktail of antibiotics
(gentamicin, metronidaxole vancmycin, and colistin at appropriate
dosages). Control mice receive a single dose of clindamycin (10
mg/kg) intraperitoneally, whereas experimental mice receive
clindamycin intraperitoneally plus 10.sup.8 CFU of the engineered
EcN variant via oral gavage. One day later, all mice are infected
by oral gavage with 0.5 ml of a suspension of C. difficile strain
VPI 10463 (5.times.10.sup.8 CFU/ml). Control mice are further given
drinking water alone, and experimental mice are also further
administered the EcN variants orally for 6 days and monitored for
weight loss and survival. Fecal samples are collected daily and
tissues are collected on day 10, and the localization of the EcN is
measure by CFU counting and immunohistochemistry.
Example X
Selective Capture of Harmful Agents, Toxins or Metabolites
[0154] Aspects of the present disclosure are directed to methods of
capturing capture targets such as harmful agents, toxins or
metabolites by administering to an individual in need thereof a
certain engineered bacterial strain or strains as described herein.
According to one aspect, the bacterial strain expresses a
CsgA-linker-capture agent polypeptide construct. Certain capture
agents and associated capture targets include cholesterol and
cholesterol-binding-peptides, phosphate and
phosphate-binding-peptides, gliadin and gliadin-binding peptides
and the like and those readily identified through literature
search. Representative examples are shown in Table 5 below.
TABLE-US-00007 Name Sequence Origin Function L4F peptide
DWFKAFYDKVAEKFKEAF Apolipoprotein E Cholesterol (SEQ ID NO: 34)
binding Alpha synuclein GGAVVTGVTAVA Alpha synuclein Cholesterol
peptide (SEQ ID NO: 35) protein binding P61 VRPMPLQ Phage Display
Gliadin binding (SEQ ID NO: 36) P64 LTHPQDSPPASA Phage Display
Gliadin binding (SEQ ID NO: 37) GlnBP LVVATDTAFVPFEFKQGDKYVGFDVDL
glutamine-binding Gliadin binding WAAIAKELKLDYELKPMDFSGIIPALQT
protein (GlnBP) KNVDALAGITITDERKKAIDFSDGYYKS
GLLVMVKANNNDVKSVKDLDGKVVAV KSGTGSVDYAKANIKTKDLRQFPNIDNA
YMELGTNRADAVLHDTPNILYFIKTAGN GQFKAVGDSLEAQYGIAFPKGSD ELRDKVNGAL
KTLRENGTYN EIYKKWFGTE PK (SEQ ID NO: 38)
[0155] The bacterial strain expresses the CsgA-linker-capture agent
polypeptide construct and the capture agent binds to the capture
target. According to one aspect, the bacterial strain expresses a
CsgA-linker-first member of a binding pair polypeptide construct.
The second member of the binding pair attached to a capture agent
is then administered. The first member of the binding pair and the
second member of the binding pair bind thereby localizing the
capture to the bacteria cell.
[0156] Useful bacterial strains include non-pathogenic E. coli,
such as the Nissle strain, MG1655, K12-derived strains and the
like.
[0157] According to one aspect, the capture agent protein or
polypeptide may exert its effects while still attached to the
engineered curli fibers. According to one aspect, the capture agent
protein or polypeptide may exert its effects as a soluble protein
after cleavage from the curli fibers by a protease. Several
proteases in the MMP family are upregulated during inflammation or
may be otherwise available in the gastrointestinal tract and may be
used to cleave the bioactive domain from the linker domain.
[0158] According to one aspect, the capture agent proteins or
peptides may be released from the curli fibers using a
protease-cleavable linker. According to this aspect, a recombinant
construct of CsgA-linker-capture agent is made where the linker
domain includes an amino acid sequence that is recognized and
cleaved by a protease enzyme. Such amino acid sequences and
associated protease enzymes are known to those of skill in the art.
The linker may be selected such that it is susceptible to cleavage
by enzymes that are produced locally at sites of inflammation
(MMP2, MMP9, etc.). Methods described above can be used to confirm
both in vitro and in vivo activity of this embodiment of the
disclosure.
Example XI
Engineered Bacteria as Live Diagnostics
[0159] Aspects of the present disclosure are directed to methods of
delivering a diagnostic agent, such as a marker, to a site within a
mammal by administering to the mammal a certain engineered
bacterial strain or strains as described herein. According to one
aspect, the bacterial strain expresses a CsgA-linker-diagnostic
polypeptide construct.
[0160] The bacterial strain expresses the CsgA-linker-diagnostic
polypeptide construct and the diagnostic polypeptide is detected.
According to one aspect, the bacterial strain expresses a
CsgA-linker-first member of a binding pair polypeptide construct.
The bacterial strains express the CsgA-linker-first member of a
binding pair polypeptide construct. The second member of the
binding pair attached to a diagnostic compound is then
administered. The first member of the binding pair and the second
member of the binding pair bind thereby localizing the diagnostic
compound to the location of interest, such as the gut.
[0161] Useful bacterial strains include non-pathogenic E. coli,
such as the Nissle strain, MG1655, K12-derived strains and the
like.
[0162] According to one aspect, the diagnostic polypeptide or
diagnostic compound may be still attached to the engineered curli
fibers. According to one aspect, the diagnostic polypeptide or the
diagnostic compound may be cleaved from the curli fibers by a
protease. Several proteases in the MMP family are upregulated
during inflammation and may be used to cleave the diagnostic
polypeptide or diagnostic compound from the linker domain.
[0163] According to one aspect, the diagnostic polypeptide or
diagnostic compound may be released from the curli fibers using a
protease-cleavable linker. According to this aspect, a recombinant
construct of CsgA-linker-diagnostic polypeptide is made where the
linker domain includes an amino acid sequence that is recognized
and cleaved by a protease enzyme. Such amino acid sequences and
associated protease enzymes are known to those of skill in the art.
The linker may be selected such that it is susceptible to cleavage
by enzymes that are produced locally at sites of inflammation
(MMP2, MMP9, etc.). Methods described above can be used to confirm
both in vitro and in vivo activity of this embodiment of the
disclosure.
Example XII
Additional Applications
[0164] Aspects of the present disclosure utilize the modified
bacterial cells which express a fusion of a CsgA protein linked to
a non-native functional polypeptide by a linker for biocatalysis.
According to this aspect, methods are provided for the use of curli
derived materials and biofilms as described herein as
functionalizable surfaces for the immobilization of enzymes, such
as enzymes used to perform chemical transformations in industrial
applications (water purification, biofuel generation, etc.) and
pharmaceutical applications (synthesis of drug intermediates). The
CsgA-linker-immobilized enzyme includes the linkers described
herein.
[0165] According to an additional aspect, methods are provided for
metal removal or recovery. According to this aspect, methods are
provided for the use of curli derived materials and biofilms as
described herein as functionalizable surfaces for the
immobilization of metals. The CsgA-linker-metal binding polypeptide
or agent includes the linkers described herein. The metal binding
polypeptide or agent may bind specific metals in ionic and metallic
form.
[0166] According to additional aspects, methods are provided for
bioremediation and for purification, such as by providing affinity
purification matrices.
[0167] The curli system described herein is a biologically produced
peptide-functionalized surface coating capable of being programmed
to specifically immobilize another chemical or biological entity or
to exhibit specific binding properties. The displayed peptide may
possess intrinsic properties such as binding to other exogenously
added functional components, such as inorganic nanoparticles
(especially those with interesting opto-electronic properties or
magneto-responsiveness), carbon-based nanostructures (i.e.,
graphene or nanotubes, which may confer conductivity), or
environmental toxins (i.e., hormones or toxic metals). The
engineered biofilms can also be used to display peptides that
template the formation of inorganic or organic materials.
Functionalizing the biofilm with peptides that specifically bind to
different materials allows the surface coating of these materials
in a genetically programmable manner. In addition, applications
whereby the living biofilm is used to immobilize and present any
arbitrary protein, as might be useful for applications in
biocatalysis, biotemplating, or biosensing are specifically
contemplated. According to one aspect, the synthesis and assembly
of the material described herein is accomplished entirely by the
bacterial cell, which acts as a factory for the production of
programmed nanomaterials.
[0168] Additional specific applications include
biologically-produced nanomaterials that have programmable optical,
magnetoresistive or semiconductor properties from either the
peptide/immobilized protein itself or by the induction of templated
materials. By displaying catalytic peptides or enzymes on the curli
biofilm, a system for high-efficiency immobilized biocatalysis in
which various immobilization substrates can be used for the
adhesion of the biofilm and which can be used in any bioreactor
design is provided. The peptide/immobilized proteins can also
encode for biologically active biomolecules that will allow the
biofilm to act as a tissue scaffold or vaccine delivery material.
Expression of peptide/immobilized proteins that bind to or
enzymatically neutralize environmental toxins such as synthetic
hormones, small molecules, or toxic metals is used as a
biofilm-based technology for bioremediation. By expressing peptides
that are able to specifically bind to precious metals such as gold,
silver, platinum, and rhodium on the biofilms described herein, an
active surface area for the profitable recovery of such precious
materials is provided. The curli nanofibers can be engineered as
conductive nanowires for numerous advanced materials applications
by the display of peptides/proteins that are inherently conductive,
or by the templating/anchoring of materials that are conductive.
The use of bacteria to generate nanowires for energy storage based
upon the expression on the curli biofilm of peptides capable of
templating conductive or semiconductive materials, such as FePO4,
is provided. Bacteria can be specifically engineered via the
displayed peptide to bind strongly to specific substrates, such as
steel, glass, or gold. Such material-specific binding can provides
a biofilm-based biosensing apparatus. The curli nanofiber matrix
can also be engineered to display peptides/proteins that interact
with other molecules in order to enhance or alter the mechanical
properties of another material. By engineering the curli to adhere
to specific materials, the biofilm can act as a living coating
capable of providing adaptive and regenerative benefits, such as
biocatalysis on a wide variety of immobilization substrates,
corrosion resistance to the material, enhanced biofilm coverage for
microbial fuel cell applications, or act as an environmentally
responsive organic (biofilm)-inorganic (substrate) material.
Sequence CWU 1
1
401151PRTEscherichia coli 1Met Lys Leu Leu Lys Val Ala Ala Ile Ala
Ala Ile Val Phe Ser Gly 1 5 10 15 Ser Ala Leu Ala Gly Val Val Pro
Gln Tyr Gly Gly Gly Gly Asn His 20 25 30 Gly Gly Gly Gly Asn Asn
Ser Gly Pro Asn Ser Glu Leu Asn Ile Tyr 35 40 45 Gln Tyr Gly Gly
Gly Asn Ser Ala Leu Ala Leu Gln Thr Asp Ala Arg 50 55 60 Asn Ser
Asp Leu Thr Ile Thr Gln His Gly Gly Gly Asn Gly Ala Asp 65 70 75 80
Val Gly Gln Gly Ser Asp Asp Ser Ser Ile Asp Leu Thr Gln Arg Gly 85
90 95 Phe Gly Asn Ser Ala Thr Leu Asp Gln Trp Asn Gly Lys Asn Ser
Glu 100 105 110 Met Thr Val Lys Gln Phe Gly Gly Gly Asn Gly Ala Ala
Val Asp Gln 115 120 125 Thr Ala Ser Asn Ser Ser Val Asn Val Thr Gln
Val Gly Phe Gly Asn 130 135 140 Asn Ala Thr Ala His Gln Tyr 145 150
21997DNAArtificialCsgA gene verification
regionmisc_feature(1)..(9)n is a, c, g, or tmisc_feature(14)..(14)n
is a, c, g, or tmisc_feature(19)..(19)n is a, c, g, or
tmisc_feature(21)..(21)n is a, c, g, or tmisc_feature(30)..(30)n is
a, c, g, or tmisc_feature(36)..(36)n is a, c, g, or
tmisc_feature(1981)..(1986)n is a, c, g, or
tmisc_feature(1989)..(1997)n is a, c, g, or t 2nnnnnnnnnc
gcancaganc nttctctccn ggttcntctt atgctcgata tttcaacaaa 60ttaagacttt
tctgaagagg gcagccattg ttgtgataaa tgaagtgact gtccatcaga
120aacagtaaca actattttca cccgatcgtc cggggaaata tttaaactca
acttcgtcaa 180agcaatgggt tgattagcag gcaatgagag ggtcttttct
tgcttcgtct gactttgccc 240tgaactgcct tcgcgcaggg acaatatttg
tactctgcac agacaagatt gagtaagagt 300gacttcagga ataatggtgt
acatatcccc ttgctgggtc gtattaaagg ttatctgact 360ggaaagtgcc
gcaaggagta ataacgcatt catattcttc tcccgaaaaa aaacagggct
420tgcgccgtgt aggctggagc tgcttcgaag ttcctatact ttctagagaa
taggaacttc 480ggaataggaa cttcatttaa atggcgcgcc ttacgccccg
ccctgccact catcgcagta 540ctgttgtatt cattaagcat ctgccgacat
ggaagccatc acaaacggca tgatgaacct 600gaatcgccag cggcatcagc
accttgtcgc cttgcgtata atatttgccc atggtgaaaa 660cgggggcgaa
gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc
720agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
taggccaggt 780tttcaccgta acacgccaca tcttgcgaat atatgtgtag
aaactgccgg aaatcgtcgt 840ggtattcact ccagagcgat gaaaacgttt
cagtttgctc atggaaaacg gtgtaacaag 900ggtgaacact atcccatatc
accagctcac cgtctttcat tgccatacgt aattccggat 960gagcattcat
caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt
1020tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
taggtacatt 1080gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg
ccattgggat atatcaacgg 1140tggtatatcc agtgattttt ttctccattt
tagcttcctt agctcctgaa aatctcgaca 1200actcaaaaaa tacgcccggt
agtgatctta tttcattatg gtgaaagttg gaacctctta 1260cgtgccgatc
aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag
1320ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtagg
cgcgccgaag 1380ttcctatact ttctagagaa taggaacttc ggaataggaa
ctaaggagga tattcatatg 1440gaccatggct aattcccatg taaaaccccc
atcggattga tttaaaagtc gaatggaaat 1500taacgttgtg tcacgcgaat
agccatttgc gactgtctct gcactacaat tgccgttttt 1560tgagtaccat
actgtgtaat atttgcttta ttaccagaac ctttctggat aatcatcgca
1620gtattaccat aagcaccttg cgaaatactg gcatcgttgg cactgcccgc
ctgatcaata 1680tatgcaaggt tataatctcc tgtctgatca atctttgccc
agttgctact accttcttgc 1740gcaacaaccg tcaaaagttt tgagcctccc
tgccgtaact gagcactatt attagtccca 1800gcttgaccaa ttatggctgc
ctgattaaat gaagacttac tcaattcatt taccgcaaag 1860ttatattctg
aattagctaa atcataacct gctgcggctg caatcccagg cgcacccagt
1920attgttaaca tcataaataa caatttgttt ttcatgttgt caccctggac
ctggtcgtac 1980nnnnnnaann nnnnnnn
1997319PRTArtificiallinkermisc_feature(16)..(19)Xaa can be any
naturally occurring amino acid 3Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser Xaa 1 5 10 15 Xaa Xaa Xaa
44PRTArtificiallinker 4Gly Gly Gly Ser 1 512PRTArtificiallinker
5Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10
624PRTArtificiallinker 6Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser 20
748PRTArtificiallinker 7Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser 35 40 45 812PRTArtificiallinker
8Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1 5 10
924PRTArtificiallinker 9Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
Pro Pro Pro Pro Pro 1 5 10 15 Pro Pro Pro Pro Pro Pro Pro Pro 20
1015PRTArtificiallinker 10Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
Glu Ala Ala Ala Lys 1 5 10 15 1145PRTArtificiallinker 11Glu Ala Ala
Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu 1 5 10 15 Ala
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala 20 25
30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 35 40 45
1280PRTArtificiallinker 12Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 35 40 45 Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 50 55 60 Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 65 70 75 80
1330PRTArtificiallinker_nucleotides 1 to 29 may be absent 13Pro Pro
Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1 5 10 15
Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 20 25 30
145PRTArtificiallinker 14Glu Ala Ala Ala Lys 1 5
1575PRTArtificiallinker_EAAAK may be absent 1 to 14 times 15Glu Ala
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu 1 5 10 15
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala 20
25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
Ala 35 40 45 Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
Ala Ala Ala 50 55 60 Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 65
70 75 1648PRTArtificiallinker 16Gly Gly Gly Ser Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 35 40 45
1724PRTArtificiallinker 17Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
Pro Pro Pro Pro Pro Pro 1 5 10 15 Pro Pro Pro Pro Pro Pro Pro Pro
20 1845PRTArtificiallinker 18Glu Ala Ala Ala Lys Glu Ala Ala Ala
Lys Glu Ala Ala Ala Lys Glu 1 5 10 15 Ala Ala Ala Lys Glu Ala Ala
Ala Lys Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala
Ala Ala Lys Glu Ala Ala Ala Lys 35 40 45 197PRTArtificialbinding
domain 19Val His Leu Gly Tyr Ala Thr 1 5 2012PRTArtificialbinding
domain 20Leu Glu Thr Thr Cys Ala Ser Leu Cys Tyr Pro Ser 1 5 10
217PRTArtificialbinding domain 21Val Arg Pro Met Pro Leu Gln 1 5
2212PRTArtificialbinding domain 22Leu Thr His Pro Gln Asp Ser Pro
Pro Ala Ser Ala 1 5 10 23106PRTArtificialbinding domain 23Glu Lys
Pro Ala Ala Cys Arg Cys Ser Arg Gln Asp Pro Lys Asn Arg 1 5 10 15
Val Asn Cys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe Thr Ser 20
25 30 Gly Cys Cys Phe Asp Ser Gln Val Pro Gly Val Pro Trp Cys Phe
Lys 35 40 45 Pro Leu Pro Ala Gln Glu Ser Glu Glu Cys Val Met Gln
Val Ser Ala 50 55 60 Arg Lys Asn Cys Gly Tyr Pro Gly Ile Ser Pro
Glu Asp Cys Ala Ala 65 70 75 80 Arg Asn Cys Cys Phe Ser Asp Thr Ile
Pro Glu Val Pro Trp Cys Phe 85 90 95 Phe Pro Met Ser Val Glu Asp
Cys His Tyr 100 105 2459PRTArtificialbinding domain 24Glu Glu Tyr
Val Gly Leu Ser Ala Asn Gln Cys Ala Val Pro Ala Lys 1 5 10 15 Asp
Arg Val Asp Cys Gly Tyr Pro His Val Thr Pro Lys Glu Cys Asn 20 25
30 Asn Arg Gly Cys Cys Phe Asp Ser Arg Ile Pro Gly Val Pro Trp Cys
35 40 45 Phe Lys Pro Leu Gln Glu Ala Glu Cys Thr Phe 50 55
2512PRTArtificialpeptide domain 25Leu Thr His Pro Gln Asp Ser Pro
Pro Ala Ser Ala 1 5 10 267PRTArtificialpeptide domain 26Val His Leu
Gly Tyr Ala Thr 1 5 2712PRTArtificialpeptide domain 27Leu Glu Thr
Thr Cys Ala Ser Leu Cys Tyr Pro Ser 1 5 10 287PRTArtificialpeptide
domain 28Val Arg Pro Met Pro Leu Gln 1 5 295PRTArtificialproline
sequencemisc_feature(3)..(3)Xaa can be any naturally occurring
amino acid 29Leu Pro Xaa Thr Gly 1 5 3019PRTArtificiallinker_GGS
repeat may be absent 3 to 4 timesmisc_feature(16)..(19)Xaa can be
any naturally occurring amino acid 30Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Xaa 1 5 10 15 Xaa Xaa Xaa
3143PRTArtificialbinding domain 31Ser Lys Trp Gln His Gln Gln Asp
Ser Cys Arg Lys Gln Leu Gln Gly 1 5 10 15 Val Asn Leu Thr Pro Cys
Glu Lys His Ile Met Glu Lys Ile Gln Gly 20 25 30 Arg Gly Asp Asp
Asp Asp Asp Asp Asp Asp Asp 35 40 32134PRTArtificialbinding domain
32Met Met Met Pro Ala Asn Tyr Ser Val Ile Ala Glu Asn Glu Met Thr 1
5 10 15 Tyr Val Asn Gly Gly Ala Asn Phe Ile Asp Ala Ile Gly Ala Val
Thr 20 25 30 Ala Pro Ile Trp Thr Leu Asp Asn Val Lys Thr Phe Asn
Thr Asn Ile 35 40 45 Val Thr Leu Val Gly Asn Thr Phe Leu Gln Ser
Thr Ile Asn Arg Thr 50 55 60 Ile Val Leu Phe Ser Gly Asn Thr Thr
Trp Lys Glu Val Gly Asn Ile 65 70 75 80 Gly Lys Asn Leu Phe Gly Thr
Asn Val Lys Gly Asn Pro Ile Glu Lys 85 90 95 Asn Asn Phe Gly Asp
Tyr Ala Met Asn Ala Leu Gly Ile Ala Ala Ala 100 105 110 Val Tyr Asn
Leu Gly Val Ala Pro Thr Lys Asn Thr Val Lys Glu Thr 115 120 125 Glu
Val Lys Phe Thr Val 130 3318PRTArtificialbinding peptide 33Asp Trp
Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu 1 5 10 15
Ala Phe 3412PRTArtificialbinding peptide 34Gly Gly Ala Val Val Thr
Gly Val Thr Ala Val Ala 1 5 10 3512PRTArtificialbinding peptide
35Gly Gly Ala Val Val Thr Gly Val Thr Ala Val Ala 1 5 10
3612PRTArtificialbinding peptide 36Leu Thr His Pro Gln Asp Ser Pro
Pro Ala Ser Ala 1 5 10 37220PRTArtificialbinding peptide 37Leu Val
Val Ala Thr Asp Thr Ala Phe Val Pro Phe Glu Phe Lys Gln 1 5 10 15
Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu Trp Ala Ala Ile Ala 20
25 30 Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro Met Asp Phe Ser
Gly 35 40 45 Ile Ile Pro Ala Leu Gln Thr Lys Asn Val Asp Ala Leu
Ala Gly Ile 50 55 60 Thr Ile Thr Asp Glu Arg Lys Lys Ala Ile Asp
Phe Ser Asp Gly Tyr 65 70 75 80 Tyr Lys Ser Gly Leu Leu Val Met Val
Lys Ala Asn Asn Asn Asp Val 85 90 95 Lys Ser Val Lys Asp Leu Asp
Gly Lys Val Val Ala Val Lys Ser Gly 100 105 110 Thr Gly Ser Val Asp
Tyr Ala Lys Ala Asn Ile Lys Thr Lys Asp Leu 115 120 125 Arg Gln Phe
Pro Asn Ile Asp Asn Ala Tyr Met Glu Leu Gly Thr Asn 130 135 140 Arg
Ala Asp Ala Val Leu His Asp Thr Pro Asn Ile Leu Tyr Phe Ile 145 150
155 160 Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala Val Gly Asp Ser Leu
Glu 165 170 175 Ala Gln Tyr Gly Ile Ala Phe Pro Lys Gly Ser Asp Glu
Leu Arg Asp 180 185 190 Lys Val Asn Gly Ala Leu Lys Thr Leu Arg Glu
Asn Gly Thr Tyr Asn 195 200 205 Glu Ile Tyr Lys Lys Trp Phe Gly Thr
Glu Pro Lys 210 215 220 3860PRTArtificialbinding domain 38Glu Ala
Gln Thr Glu Thr Cys Thr Val Ala Pro Arg Glu Arg Gln Asn 1 5 10 15
Cys Gly Phe Pro Gly Val Thr Pro Ser Gln Cys Ala Asn Lys Gly Cys 20
25 30 Cys Phe Asp Asp Thr Val Arg Gly Val Pro Trp Cys Phe Tyr Pro
Asn 35 40 45 Thr Ile Asp Val Pro Pro Glu Glu Glu Cys Glu Phe 50 55
60 3948PRTArtificialFusion domain linker_GGGS repeat can be 48, 36,
or 24 AA 39Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser 35 40 45 4036PRTArtificiallinker_GGGS
repeat can be 36, 24, or 12 AA 40Gly Gly Gly Ser Gly Gly Gly Ser
Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser
35
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