US2014011259A1PendingUtilityA1
Protein Variants Having Modified Immunogenicity
Est. expiryApr 28, 2020(expired)· nominal 20-yr term from priority
A61K 39/00C12N 9/54A21D 2/267A21D 8/042C07K 1/047C07K 5/1008C07K 5/101C07K 5/1013C07K 5/1019C07K 5/1021C07K 14/001C07K 16/4283C11D 3/38627C11D 3/38645C12N 9/0061C12N 9/20C12Y 110/03002C07K 16/40Y10S435/911C12Y 304/21062
68
PatentIndex Score
0
Cited by
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References
0
Claims
Abstract
The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein comprising the steps obtaining antibody binding peptide sequences, using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein, defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, changing one or more of the amino acids defining the epitope area of the parent protein by genetical engineering mutations of a DNA sequence encoding the parent protein, introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and evaluating the immunogenicity of the protein variant using the parent protein as reference. The invention further relates to the protein variant and use thereof, as well as to a method for producing said protein variant.
Claims
exact text as granted — not AI-modified1 - 134 . (canceled)
135 . A protease variant having modified immunogenicity as compared to a parent protease, obtainable by a method comprising the steps of:
(a) obtaining antibody binding peptide sequences, (b) using the sequences to localise epitope sequences on the 3-dimensional structure of the parent protein, (c) defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, (d) changing one or more of the amino acids defining the epitope area of the parent protein by genetic engineering mutations of a DNA sequence encoding the parent protein, (e) introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and (f) evaluating the immunogenicity of the protein variant using the parent protein as reference.
136 . The protease variant of claim 135 , wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position
−6
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
−5
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
−4
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
−2
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
3a
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
28a
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
44a
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
44b
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
139
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
148
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
149
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion;
Position
264a
to
A, C, D, E, F, G, H, I, K,
L, M, N, P, Q, R, S, T, V,
W, Y, insertion, deletion.
137 . The protease variant of claim 135 , wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position
−1
to
G, V, L, I, W, P, C, M,
F, N, Q, Y, S, T, D, E, R, H;
Position
1
to
V, L, I, W, M, F, Y, S, T, R;
Position
2
to
G, V, I, M, F, N, Q, Y, S,
T, H;
Position
3
to
W, M, F, N, Q, Y, S, D, E,
R, H;
Position
4
to
V, L, W, M, F, Y, R;
Position
5
to
V, L, I, W, M, F, N, Q, Y,
T, R, H;
Position
6
to
G, V, L, I, W, P, M, N, Q,
T, D, E, R, H;
Position
9
to
G, V, L, I, W, P, M, F, Q,
Y, S, T, R, H;
Position
10
to
G, A, V, I, W, P, M, N, Q,
Y, S, T, D, E, R;
Position
12
to
G, A, V, L, I, W, M, F, N,
Q, Y, S, T, D, E;
Position
14
to
V, L, I, W, P, M, F, N, Q,
Y, T, R, H;
Position
15
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, E, H;
Position
17
to
G, A, V, I, W, P, M, F, Y, H;
Position
18
to
G, A, L, I, W, P, M, F, N,
Q, Y, T, D, E, H;
Position
19
to
A, V, I, W, M, F, N, Y, S,
T, D, R, H;
Position
20
to
G, V, L, I, W, M, F, N, Q,
Y, S, T, D, E;
Position
21
to
G, V, I, W, N, Q, Y, S, T,
D, E, R, H;
Position
22
to
G, V, L, I, W, M, F, Y, S, T;
Position
24
to
G, V, L, I, W, M, F, N, Q,
Y, S, D, E, R;
Position
25
to
G, A, V, L, I, W, M, F, N,
Q, Y, S, T, D, E, R, H;
Position
27
to
G, L, I, W, P, M, F, Y, T, H;
Position
38
to
V, L, I, W, M, F, N, Q, Y,
T, H;
Position
39
to
G, A, V, L, I, W, M, F, N,
Q, Y, T, D, E, R, H;
Position
40
to
V, L, I, W, M, F, N, Q, Y,
T, R, H;
Position
42
to
G, A, L, W, C, M, F, N, Q,
Y, S, T, D, E, R, H;
Position
43
to
G, L, H;
Position
44
to
G, V, L, I, W, P, M, F, Y,
S, T;
Position
45
to
G, V, L, I, W, P, M, F, N,
Q, Y, S, T, D, E, R, H;
Position
46
to
G, A, L, I, W, P, M, F, Y, H;
Position
47
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, H;
Position
48
to
A, L, I, P, M, F, N, Y, D, H;
Position
49
to
G, A, V, I, W, P, M, F, N,
Q, Y, S, T, D, E, R, H;
Position
50
to
G, A, W, M, N, Q, Y, S, T,
D, E, H;
Position
51
to
V, L, I, W, M, F, N, Y, R;
Position
52
to
V, L, I, W, M, F, Y, S,
T, R;
Position
53
to
A, V, L, I, W, M, F, N, Q,
Y, S, D, E, H;
Position
54
to
V, L, I, W, M, F, S, R;
Position
55
to
G, A, V, L, I, W, C, M, F,
N, Q, Y, T, D, E, R, K, H;
Position
56
to
G, V, L, I, W, M, F, N, Q,
Y, S, T, H;
Position
57
to
G, A, V, L, I, W, M, F, N,
Q, Y, S, T, D, E, R, K, H;
Position
58
to
L, W, M, F, N, Y, R;
Position
59
to
A, V, L, I, C, T, H;
Position
61
to
V, L, I, W, M, F, Y;
Position
62
to
G, A, L, W, M, F, N, Y, R;
Position
64
to
G, V, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
75
to
L;
Position
79
to
I;
Position
80
to
G;
Position
87
to
A, V, L, I, W, M, F, Q, Y,
S, T, D, E, H;
Position
89
to
G, V, L, I, W, P, F, N, Y,
T, E;
Position
91
to
G, A, V, L, I, W, P, M, N,
Y, S, T, D, E, R, H;
Position
98
to
A;
Position
99
to
V, L, I, W, M, F, Q, Y, H;
Position
100
to
G, V, L, I, W, M, F, Y, R, H;
Position
101
to
V, I, W, M, F, N, Q, Y, H;
Position
102
to
V, L, I, W, M, F, Y, R, H, G;
Position
108
to
I;
Position
109
to
N;
Position
112
to
E;
Position
113
to
W;
Position
115
to
I;
Position
117
to
N;
Position
118
to
N;
Position
126
to
L;
Position
127
to
G, A, V, I, W, M, F, Y, R,
H, L;
Position
128
to
I, W;
Position
129
to
W;
Position
130
to
W, F, Y, R;
Position
131
to
W, Y, R;
Position
132
to
L, W, M, F, Y, S, H;
Position
133
to
A, L, I, W, M, F, Y, R;
Position
134
to
L, I, W, F, N, Q, Y, R, H;
Position
136
to
G, A, W, P, N, Y, S, T, D,
E, H;
Position
137
to
G, A, V, I, W, P, M, N, Y, H;
Position
140
to
G, A, V, L, I, W, P, M, F, N,
Q, Y, S, T, H;
Position
141
to
G, V, L, I, W, P, M, F, Q, S,
D, E, H;
Position
143
to
V, L, I, P, M, F, N, Y, R;
Position
144
to
L, W, P, M, F, N, Q, Y, S, D,
E, R, H;
Position
145
to
G, V, L, I, W, M, F, Q, Y, D,
E, R, H;
Position
146
to
G, A, W, L, I, W, M, F, N, Q,
Y, T, D, E, R, H;
Position
155
to
V, L, I, W, M, F, Y, R;
Position
156
to
V, I, W, F, R;
Position
157
to
G, A, V, L, I, W, M, F, Y, T,
R, H;
Position
158
to
V, L, I, W, M, F, Y;
Position
159
to
A, W, M, Y, T, R, H;
Position
160
to
W, M, F, Y, R, H;
Position
161
to
I, W, M, F, Y, H;
Position
167
to
R, K;
Position
171
to
D;
Position
172
to
G, A, V, L, I, S, T, H;
Position
173
to
G, A, V, L, I, W, M, F, N, Q,
Y, S, T, E, H;
Position
181
to
G, A, V, L, I, W, C, M, F, Q,
Y, T, D, R, K, H;
Position
182
to
A, V, L, I, W, C, M, F, N, Q,
Y, S, T, D, E, H;
Position
183
to
G, A, V, L, W, C, M, F, N, Q,
Y, S, T, E, R, H;
Position
184
to
A, V, L, I, W, C, M, F, N, Q,
Y, T, E, H;
Position
185
to
G, A, V, L, I, W, C, M, F, N,
Q, Y, T, E, H;
Position
186
to
G, A, V, L, W, M, F, N, Q, Y,
S, T, D, E, R, H;
Position
188
to
G, A, V, L, W, F, S, R, K;
Position
189
to
W, F;
Position
191
to
A, V, L, I, W, M, F, Y, T,
R, H;
Position
192
to
G, L, I, W, M, N, Q, Y, S, T,
D, R, H;
Position
194
to
W, N, Q, Y, D, H;
Position
195
to
W, P, Y;
Position
196
to
G, A, V, L, I, W, P, M, F, N,
Q, Y, S, T, D, E, R, H;
Position
203
to
V, F, Y, R, H;
Position
204
to
I, W, M, Y, H;
Position
206
to
F;
Position
209
to
Y, R;
Position
210
to
W, F, Y;
Position
211
to
L, W, M, F, Y, H;
Position
212
to
V, L, I, W, M, F, Y, T, R, H;
Position
214
to
W, Y, R;
Position
215
to
A, L, I, W, M, F, Y;
Position
216
to
A, L, I, W, M, F, Y, R;
Position
217
to
W, R;
Position
218
to
G, A, L, W, P, M, F, Y, R, H;
Position
221
to
S;
Position
236
to
S;
Position
240
to
N;
Position
241
to
W;
Position
243
to
N;
Position
245
to
Q;
Position
247
to
G, V, I, W, P, F, Y, S, T, R;
Position
248
to
W, P, F, Y, E, R, H;
Position
249
to
L, W, P, F, S, D, E, H;
Position
251
to
G, L, I, W, P, M, F, Y, H;
Position
252
to
G, A, W, P, N, Q, Y, T, E, R,
H;
Position
254
to
G, V, L, I, W, M, F, N, Q,
Y, S, D, E, R, H;
Position
255
to
G, L, W, M, F, N, Y, T, D,
H;
Position
256
to
G, A, V, L, I, W, M, F, Q,
Y, S, T, D, H;
Position
257
to
G, A, L, I, W, C, M, F, N,
Q, Y, S, T, D, E, K, H;
Position
258
to
G, A, V, L, I, W, C, M, F,
N, Q, Y, S, T, E, K, H;
Position
259
to
A, V, I, W, M, F, N, Q, Y,
S, T, E, R;
Position
260
to
L, I, W, M, F, Y, T, H;
Position
261
to
L, N, S, H;
Position
262
to
G, A, V, L, I, W, P, F, N,
Q, Y, T, D, E, R, H;
Position
263
to
G, A, V, L, I, P, C, M, N,
Q, Y, S, T, R, K;
Position
265
to
V, L, I, W, M, F, Y;
Position
269
to
G, A, V, L, I, W, M, F, N,
Q, Y, S, T, E, R, H;
Position
271
to
A, L, I, W, P, M, F, N, Y,
S, T, R, H;
Position
272
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, T, D, E, H;
Position
275
to
G, A, V, L, I, W, M, F, N,
Y, T, D.
138 . The protease variant of claim 135 , wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position
−1
to
Deletion;
Position
9
to
Insertion, deletion;
Position
10
to
Insertion, deletion;
Position
12
to
Insertion, deletion;
Position
14
to
Insertion, deletion;
Position
15
to
Insertion, deletion;
Position
17
to
Insertion, deletion;
Position
18
to
Insertion, deletion;
Position
19
to
Insertion, deletion;
Position
20
to
Insertion, deletion;
Position
21
to
Insertion, deletion;
Position
22
to
Insertion, deletion;
Position
24
to
Insertion, deletion;
Position
25
to
Insertion, deletion;
Position
46
to
Insertion, deletion;
Position
47
to
Insertion, deletion;
Position
48
to
Insertion, deletion;
Position
49
to
Insertion, deletion;
Position
50
to
Insertion, deletion;
Position
51
to
Insertion, deletion;
Position
52
to
Insertion, deletion;
Position
53
to
Insertion, deletion;
Position
54
to
Insertion, deletion;
Position
55
to
Insertion, deletion;
Position
58
to
Insertion, deletion;
Position
59
to
Insertion, deletion;
Position
61
to
Insertion, deletion;
Position
64
to
Insertion, deletion;
Position
78
to
Insertion:
Position
80
to
Insertion;
Position
91
to
Insertion, deletion;
Position
98
to
Deletion;
Position
99
to
Deletion;
Position
102
to
Deletion;
Position
105
to
Insertion;
Position
108
to
Insertion;
Position
109
to
Insertion;
Position
112
to
Insertion;
Position
113
to
Insertion;
Position
115
to
Insertion;
Position
116
to
Insertion;
Position
117
to
Insertion;
Position
118
to
Insertion;
Position
131
to
Deletion;
Position
134
to
Insertion, deletion;
Position
136
to
Insertion, deletion;
Position
137
to
Insertion, deletion;
Position
140
to
Insertion, deletion;
Position
141
to
Insertion, deletion;
Position
143
to
Insertion, deletion;
Position
144
to
Insertion, deletion;
Position
145
to
Insertion, deletion;
Position
146
to
Insertion, deletion;
Position
171
to
Deletion;
Position
172
to
Deletion;
Position
173
to
Deletion;
Position
181
to
Deletion;
Position
182
to
Deletion;
Position
183
to
Deletion;
Position
184
to
Deletion;
Position
185
to
Deletion;
Position
186
to
Deletion;
Position
188
to
Deletion;
Position
189
to
Deletion;
Position
191
to
Deletion;
Position
192
to
Deletion;
Position
195
to
Deletion;
Position
196
to
Insertion, deletion;
Position
221
to
Insertion;
Position
236
to
Insertion;
Position
237
to
Insertion;
Position
238
to
Insertion;
Position
239
to
Insertion;
Position
240
to
Insertion;
Position
241
to
Insertion;
Position
242
to
Insertion;
Position
243
to
Insertion;
Position
244
to
Insertion;
Position
245
to
Insertion;
Position
247
to
Insertion, deletion;
Position
248
to
Insertion, deletion;
Position
249
to
Insertion, deletion;
Position
251
to
Insertion, deletion;
Position
252
to
Insertion, deletion;
Position
254
to
Insertion, deletion;
Position
255
to
Insertion, deletion;
Position
256
to
Insertion, deletion;
Position
257
to
Insertion, deletion;
Position
258
to
Insertion, deletion;
Position
259
to
Insertion, deletion;
Position
260
to
Insertion, deletion;
Position
261
to
Insertion, deletion;
Position
262
to
Insertion, deletion;
Position
263
to
Insertion, deletion;
Position
265
to
Insertion, deletion;
Position
269
to
Insertion, deletion;
Position
271
to
Insertion, deletion;
Position
272
to
Insertion, deletion;
Position
275
to
Insertion, deletion.
139 . The protease variant of claim 135 , wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position
7
to
G, A, V, L, I, W, P, M,
F, N, Q, Y, S, T, D, E, R, H;
Position
8
to
G, A, L, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
13
to
G, L, I, W, P, M, F, N,
Q, Y, S, D, E, H;
Position
16
to
G, A, V, L, I, W, P, M,
F, N, Q, Y, S, D, E, R, H;
Position
23
to
G, A, V, L, I, W, M, F,
Y, E, R, H;
Position
26
to
G, A, V, L, I, W, M, F,
N, Q, Y, S, T, D, E, R, H;
Position
28
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
29
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
33
to
V, L, I, W, C, M, F, N,
Q, Y, R, H;
Position
35
to
G, A, V, L, I, W, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
36
to
V, L, I, W, P, M, F, N,
Y, S, T, R, H;
Position
37
to
L, I, W, M, F, N, Q, Y,
S, R, H;
Position
41
to
G, V, L, I, W, M, F, N,
Q, Y, S, T, R, H;
Position
60
to
G, A, V, L, I, W, C, M,
F, Q, Y, T, D, R, K, H;
Position
63
to
G, A, V, L, I, W, M, F,
Y, T, R, H;
Position
73
to
A;
Position
74
to
A;
Position
81
to
V;
Position
82
to
L;
Position
86
to
G, A, V, L, I, W, M, F, N,
Q, Y, T, D, E, R, H;
Position
88
to
A, V, L, I, W, M, F, N, Q,
Y, S, T, D, E, R, H;
Position
92
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
93
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
94
to
G, V, L, I, W, P, M, F, N,
Y, T, D, E, K, H;
Position
96
to
L, W, F, Y, R, K;
Position
97
to
V, L, W, C, M, F, Y, H;
Position
111
to
I;
Position
114
to
A;
Position
119
to
M;
Position
124
to
M;
Position
135
to
G, L, P, C, N, Q, T, R, H;
Position
138
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, H;
Position
142
to
G, A, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
147
to
G, A, V, L, W, M, F, N, Q,
Y, S, T, D, E, R, K, H;
Position
151
to
G, V, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
162
to
I, W, F, Y, R;
Position
163
to
V, W, M, F, H;
Position
168
to
G, V, L, I, W, C, M, F, N,
Q, Y, S, T, D, E, R, K, H;
Position
169
to
C, E, F, G, H, I, K, L, M,
N, Q, R, T, V, W, Y;
Position
174
to
G, A, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
176
to
G, A, V, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
179
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
187
to
A, V, L, I, W, M, F, Y, R;
Position
190
to
G, A, V, L, I, W, C, M, F,
N, Q, Y, S, T, R, K, H;
Position
193
to
G, V, L, I, W, M, F, N, Q,
Y, S, T, D, E, R, H;
Position
197
to
G, V, L, I, W, P, M, F, Q,
Y, S, T, H;
Position
198
to
G, A, L, I, W, P, C, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
205
to
W, F, Y, R, K;
Position
208
to
A, V, L, I, W, C, M, F, Y,
T, R, K, H;
Position
219
to
G, A, V, L, I, W, F, Y, R, H;
Position
222
to
M;
Position
232
to
A;
Position
233
to
L;
Position
234
to
I;
Position
250
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, H;
Position
267
to
G, A, V, L, I, W, M, F, N,
Q, Y, S, T, D, E, R, H;
Position
268
to
G, V, L, I, W, C, M, N, Q,
Y, S, T, D, E, R, K, H;
Position
270
to
G, L, I, W, P, M, F, N, Q,
Y, S, T, D, E, R, K, H;
Position
273
to
G, A, V, L, I, W, P, M, F,
N, Q, Y, S, T, D, E, R, K, H;
Position
274
to
W, P, M, F, N, Q, Y, T, D,
E, R, H.
140 . The protease variant of claim 135 , wherein the protease is a subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position
13
to
Insertion, deletion;
Position
16
to
Insertion, deletion;
Position
23
to
Insertion, deletion;
Position
26
to
Insertion, deletion;
Position
28
to
Insertion, deletion;
Position
29
to
Insertion, deletion;
Position
35
to
Deletion;
Position
60
to
Insertion, deletion;
Position
63
to
Insertion;
Position
81
to
Insertion;
Position
82
to
Insertion;
Position
92
to
Insertion, deletion;
Position
93
to
Insertion, deletion;
Position
94
to
Insertion, deletion;
Position
96
to
Deletion,
Position
106
to
Insertion,
Position
111
to
Insertion,
Position
114
to
Insertion,
Position
119
to
Insertion,
Position
124
to
Insertion,
Position
138
to
Insertion, deletion;
Position
142
to
Insertion, deletion;
Position
147
to
Insertion, deletion;
Position
151
to
Insertion, deletion;
Position
174
to
Insertion, deletion;
Position
176
to
Insertion, deletion;
Position
179
to
Insertion, deletion;
Position
187
to
Deletion;
Position
190
to
Deletion;
Position
193
to
Deletion;
Position
197
to
Insertion, deletion;
Position
198
to
Insertion, deletion;
Position
232
to
Insertion,
Position
233
to
Insertion,
Position
234
to
Insertion,
Position
246
to
Insertion,
Position
250
to
Insertion, deletion;
Position
267
to
Insertion, deletion;
Position
268
to
Insertion, deletion;
Position
270
to
Insertion, deletion;
Position
273
to
Insertion, deletion.
141 . The protease variant of claim 135 , wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position 2 to
G, V, I, M, F, N, Q, Y, S, T, H,
Position 3 to
W, M, F, N, Q, Y, S, D, E, R, H,
Position 4 to
V, L, W, M, F, Y, R,
Position 6 to
G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 9 to
G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion,
Position 10 to
G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion,
Position 12 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 14 to
V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion,
deletion,
Position 17 to
G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to
G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 19 to
A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 20 to
G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 21 to
G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 22 to
G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 24 to
G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion, deletion,
Position 25 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 27 to
G, L, I, W, P, M, F, Y, T, H,
Position 37 to
L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 42 to
G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H,
Position 43 to
G, L, H,
Position 44 to
G, V, L, I, W, P, M, F, Y, S, T,
Position 45 to
G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 46 to
G, A, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 47 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 48 to
A, L, I, P, M, F, N, Y, D, H, insertion, deletion,
Position 50 to
G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 51 to
V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 54 to
V, L, I, W, M, F, S, R, deletion, insertion,
Position 55 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion,
insertion,
Position 57 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 58 to
L, W, M, F, N, Y, R, insertion, deletion,
Position 59 to
A, V, L, I, C, T, H, insertion, deletion,
Position 61 to
V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to
G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 75 to
L,
Position 78 to
insertion,
Position 79 to
I,
Position 87 to
A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 89 to
G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to
G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion,
deletion,
Position 98 to
A, deletion,
Position 100 to
G, V, L, I, W, M, F, Y, R, H,
Position 101 to
V, I, W, M, F, N, Q, Y, H,
Position 102 to
V, L, I, W, M, F, Y, R, H, G, deletion,
Position 109 to
N, insertion,
Position 112 to
E, insertion,
Position 113 to
W, insertion,
Position 116 to
insertion,
Position 117 to
N, insertion,
Position 126 to
L,
Position 127 to
G, A, V, I, W, M, F, Y, R, H, L,
Position 128 to
I, W,
Position 129 to
W,
Position 130 to
W, F, Y, R,
Position 131 to
W, Y, R, deletion,
Position 132 to
L, W, M, F, Y, S, H,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 134 to
L, I, W, F, N, Q, Y, R, H, insertion, deletion,
Position 136 to
G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 140 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion,
Position 141 to
G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 143 to
V, L, I, P, M, F, N, Y, R, insertion, deletion,
Position 144 to
L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to
G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to
G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion, deletion,
Position 155 to
V, L, I, W, M, F, Y, R,
Position 156 to
V, I, W, F, R,
Position 157 to
G, A, V, L, I, W, M, F, Y, T, R, H,
Position 158 to
V, L, I, W, M, F, Y,
Position 160 to
W, M, F, Y, R, H,
Position 161 to
I, W, M, F, Y, H,
Position 167 to
R, K,
Position 170 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 171 to
D, deletion,
Position 172 to
G, A, V, L, I, S, T, H, deletion,
Position 173 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to
G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 183 to
G, A, V, L, W, C, M, F, N, Q, Y, S, T, E, R, H, deletion,
Position 184 to
A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 185 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to
G, A, V, L, W, F, S, R, K, deletion,
Position 189 to
W, F, deletion,
Position 191 to
A, V, L, I, W, M, F, Y, T, R, H, deletion,
Position 192 to
G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 195 to
W, P, Y, deletion,
Position 197 to
G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 203 to
V, F, Y, R, H,
Position 206 to
F,
Position 209 to
Y, R,
Position 210 to
W, F, Y,
Position 212 to
V, L, I, W, M, F, Y, T, R, H,
Position 214 to
W, Y, R,
Position 216 to
A, L, I, W, M, F, Y, R,
Position 217 to
W, R,
Position 218 to
G, A, L, W, P, M, F, Y, R, H,
Position 221 to
S, insertion,
Position 236 to
S, insertion,
Position 237 to
insertion,
Position 239 to
insertion,
Position 240 to
N, insertion,
Position 241 to
W, insertion,
Position 242 to
insertion,
Position 244 to
insertion,
Position 245 to
Q, insertion,
Position 247 to
G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 248 to
W, P, F, Y, E, R, H, insertion, deletion,
Position 251 to
G, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 252 to
G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 255 to
G, L, W, M, F, N, Y, T, D, H, insertion, deletion,
Position 256 to
G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion,
Position 257 to
G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion,
deletion,
Position 258 to
G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion,
deletion,
Position 259 to
A, V, I, W, M, F, N, Q, Y, S, T, E, R, insertion, deletion,
Position 260 to
L, I, W, M, F, Y, T, H, insertion, deletion,
Position 261 to
L, N, S, H, insertion, deletion,
Position 262 to
G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 263 to
G, A, V, L, I, P, C, M, N, Q, Y, S, T, R, K, insertion,
deletion,
Position 265 to
V, L, I, W, M, F, Y, insertion, deletion,
Position 271 to
A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion,
Position 272 to
G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion,
deletion,
Position 275 to
G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.
142 . The protease variant of claim 141 , wherein the savinase-like subtilisin comprises one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position 6 to
G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 9 to
G, V, L, I, W, P, M, F, Q, Y, S, T, R, H, insertion, deletion,
Position 10 to
G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion, deletion,
Position 14 to
V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion,
deletion,
Position 17 to
G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to
G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion, deletion,
Position 19 to
A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 20 to
G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion, deletion,
Position 21 to
G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 37 to
L, I, W, M, F, N, Q, Y, S, R, H,
Position 43 to
G, L, H,
Position 45 to
G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 47 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 50 to
G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 51 to
V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 54 to
V, L, I, W, M, F, S, R, deletion, insertion,
Position 59 to
A, V, L, I, C, T, H, insertion, deletion,
Position 89 to
G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to
G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion,
deletion,
Position 101 to
V, I, W, M, F, N, Q, Y, H,
Position 109 to
N, insertion,
Position 112 to
E, insertion,
Position 113 to
W, insertion,
Position 127 to
G, A, V, I, W, M, F, Y, R, H, L,
Position 128 to
I, W,
Position 129 to
W,
Position 130 to
W, F, Y, R,
Position 131 to
W, Y, R, deletion,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 136 to
G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 140 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, H, insertion, deletion,
Position 141 to
G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 143 to
V, L, I, P, M, F, N, Y, R, insertion, deletion,
Position 144 to
L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to
G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to
G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 155 to
V, L, I, W, M, F, Y, R,
Position 157 to
G, A, V, L, I, W, M, F, Y, T, R, H,
Position 158 to
V, L, I, W, M, F, Y,
Position 160 to
W, M, F, Y, R, H,
Position 161 to
I, W, M, F, Y, H,
Position 167 to
R, K,
Position 170 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 171 to
D, deletion,
Position 172 to
G, A, V, L, I, S, T, H, deletion,
Position 173 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to
G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 184 to
A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 185 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to
G, A, V, L, W, F, S, R, K, deletion,
Position 189 to
W, F, deletion,
Position 192 to
G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 195 to
W, P, Y, deletion,
Position 197 to
G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 203 to
V, F, Y, R, H,
Position 210 to
W, F, Y,
Position 218 to
G, A, L, W, P, M, F, Y, R, H,
Position 236 to
S, insertion,
Position 237 to
insertion,
Position 239 to
insertion,
Position 240 to
N, insertion,
Position 241 to
W, insertion,
Position 242 to
insertion,
Position 244 to
insertion,
Position 245 to
Q, insertion,
Position 247 to
G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 251 to
G, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 255 to
G, L, W, M, F, N, Y, T, D, H, insertion, deletion,
Position 256 to
G, A, V, L, I, W, M, F, Q, Y, S, T, D, H, insertion, deletion,
Position 257 to
G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion,
deletion,
Position 258 to
G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, E, K, H, insertion,
deletion,
Position 260 to
L, I, W, M, F, Y, T, H, insertion, deletion,
Position 262 to
G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 265 to
V, L, I, W, M, F, Y, insertion, deletion,
Position 271 to
A, L, I, W, P, M, F, N, Y, S, T, R, H, insertion, deletion,
Position 272 to
G, A, V, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion,
deletion,
Position 275 to
G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion.
143 . The savinase-like subtilisin of claim 141 , wherein the subtilisin has at least 81% homology to SEQ ID NO: 24.
144 . The savinase-like subtilisin of claim 141 , wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.
145 . The protein variant of claim 135 , wherein the protease is a savinase-like subtilisin comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position 8 to
G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 16 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 23 to
G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,
Position 26 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 35 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H, deletion,
Position 38 to
V, L, I, W, M, F, N, Q, Y, T, H,
Position 39 to
G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 41 to
G, V, L, I, W, M, F, N, Q, Y, S, T, R, H,
Position 60 to
G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, insertion,
deletion,
Position 73 to
A,
Position 74 to
A,
Position 80 to
G, insertion,
Position 81 to
V, insertion,
Position 86 to
G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 88 to
A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
Position 90 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 93 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 108 to
I, insertion,
Position 111 to
I, insertion,
Position 124 to
M, insertion,
Position 135 to
G, L, P, C, N, Q, T, R, H,
Position 142 to
G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 147 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 148 to
G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 149 to
G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 151 to
G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 163 to
V, W, M, F, H,
Position 168 to
G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 169 to
C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y,
Position 174 to
G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 179 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 190 to
G, A, V, L, I, W, C, M, F, N, Q, Y, S, T, R, K, H, deletion,
Position 193 to
G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 196 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 208 to
A, V, L, I, W, C, M, F, Y, T, R, K, H,
Position 213 to
N, oN, E,
Position 215 to
A, L, I, W, M, F, Y,
Position 232 to
A, insertion,
Position 233 to
L, insertion,
Position 234 to
I, insertion,
Position 246 to
insertion,
Position 250 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 254 to
G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 267 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 268 to
G, V, L, I, W, C, M, N, Q, Y, S, T, D, E, R, K, H, insertion,
deletion,
Position 269 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, R, H, insertion,
deletion,
Position 273 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion.
146 . The savinase-like subtilisin of claim 145 , wherein the subtilisin has at least 81% homology to SEQ ID NO: 24.
147 . The savinase-like subtilisin of claim 146 , wherein the subtilisin has any of the amino acid sequence of SEQ ID NO: 24, 26, 27, 28, 29, 30, 31, 32, 34, 35.
148 . The protease variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 25 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 25:
Position 21 to
G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 27 to
G, L, I, W, P, M, F, Y, T, H,
Position 50 to
G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 52 to
V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 55 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H, deletion,
insertion,
Position 129 to
W,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 172 to
G, A, V, L, I, S, T, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 195 to
W, P, Y, deletion,
Position 197 to
G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 242 to
insertion,
Position 249 to
L, W, P, F, S, D, E, H, insertion, deletion,
Position 252 to
G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 254 to
G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 257 to
G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion,
deletion,
Position 260 to
L, I, W, M, F, Y, T, H, insertion, deletion,
Position 265 to
V, L, I, W, M, F, Y, insertion, deletion,
with the proviso that the amino acids of the parent enzyme are substituted
to another amino acid.
149 . The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 10 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 10:
Position 4 to
V, L, W, M, F, Y, R,
Position 38 to
V, L, I, W, M, F, N, Q, Y, T, H,
Position 40 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 43 to
G, L, H,
Position 47 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 49 to
G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 54 to
V, L, I, W, M, F, S, R, deletion, insertion,
Position 96 to
L, W, F, Y, R, K, deletion,
Position 99 to
V, L, I, W, M, F, Q, Y, H, deletion,
Position 113 to
W, insertion,
Position 131 to
W, Y, R, deletion,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 141 to
G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion,
deletion,
Position 144 to
L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 170 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
Position 173 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, E, H, deletion,
Position 181 to
G, A, V, L, I, W, C, M, F, Q, Y, T, D, R, K, H, deletion,
Position 185 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, E, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to
G, A, V, L, W, F, S, R, K, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 203 to
V, F, Y, R, H,
Position 210 to
W, F, Y,
Position 211 to
L, W, M, F, Y, H,
Position 257 to
G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H, insertion,
deletion,
Position 261 to
L, N, S, H, insertion, deletion,
Position 262 to
G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 265 to
V, L, I, W, M, F, Y, insertion, deletion.
with the proviso that the amino acids of the parent enzyme are substituted
to another amino acid.
150 . The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 11 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 11:
Position 38 to
V, L, I, W, M, F, N, Q, Y, T, H,
Position 40 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 45 to
G, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 47 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 49 to
G, A, V, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 50 to
G, A, W, M, N, Q, Y, S, T, D, E, H, insertion, deletion,
Position 52 to
V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 53 to
A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion,
insertion,
Position 56 to
G, V, L, I, W, M, F, N, Q, Y, S, T, H,
Position 58 to
L, W, M, F, N, Y, R, insertion, deletion,
Position 96 to
L, W, F, Y, R, K, deletion,
Position 97 to
V, L, W, C, M, F, Y, H,
Position 98 to
A, deletion,
Position 105 to
insertion,
Position 109 to
N, insertion,
Position 113 to
W, insertion,
Position 115 to
I, insertion,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 136 to
G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 141 to
G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion,
deletion,
Position 158 to
V, L, I, W, M, F, Y,
Position 159 to
A, W, M, Y, T, R, H,
Position 172 to
G, A, V, L, I, S, T, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H,
deletion,
Position 189 to
W, F, deletion,
Position 192 to
G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 195 to
W, P, Y, deletion,
Position 197 to
G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion,
deletion,
Position 257 to
G, A, L, I, W, C, M, F, N, Q, Y, S, T, D, E, K, H,
insertion, deletion,
Position 261 to
L, N, S, H, insertion, deletion,
Position 265 to
V, L, I, W, M, F, Y, insertion, deletion,
with the proviso that the amino acids of the parent enzyme are substituted
to another amino acid.
151 . The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33:
Position −6 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y,
insertion, deletion,
Position −5 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, Y,
insertion, deletion,
Position −4 to
A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y,
insertion, deletion,
Position −2 to
A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position −1 to
G, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 1 to
V, L, I, W, M, F, Y, S, T, R,
Position 2 to
G, V, I, M, F, N, Q, Y, S, T, H,
Position 3a to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 5 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 6 to
G, V, L, I, W, P, M, N, Q, T, D, E, R, H,
Position 7 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
Position 8 to
G, A, L, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 10 to
G, A, V, I, W, P, M, N, Q, Y, S, T, D, E, R, insertion,
deletion,
Position 12 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, insertion,
deletion,
Position 13 to
G, L, I, W, P, M, F, N, Q, Y, S, D, E, H, insertion,
deletion,
Position 14 to
V, L, I, W, P, M, F, N, Q, Y, T, R, H, insertion, deletion,
Position 15 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, E, H, insertion,
deletion,
Position 16 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 17 to
G, A, V, I, W, P, M, F, Y, H, insertion, deletion,
Position 18 to
G, A, L, I, W, P, M, F, N, Q, Y, T, D, E, H, insertion,
deletion,
Position 19 to
A, V, I, W, M, F, N, Y, S, T, D, R, H, insertion, deletion,
Position 21 to
G, V, I, W, N, Q, Y, S, T, D, E, R, H, insertion, deletion,
Position 22 to
G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 23 to
G, A, V, L, I, W, M, F, Y, E, R, H, insertion, deletion,
Position 24 to
G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, insertion,
deletion,
Position 25 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 26 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 27 to
G, L, I, W, P, M, F, Y, T, H,
Position 28 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 28a to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 29 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 33 to
V, L, I, W, C, M, F, N, Q, Y, R, H,
Position 35 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
deletion,
Position 37 to
L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 42 to
G, A, L, W, C, M, F, N, Q, Y, S, T, D, E, R, H,
Position 43 to
G, L, H,
Position 44 to
G, V, L, I, W, P, M, F, Y, S, T,
Position 44a to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 44b to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 46 to
G, A, L, I, W, P, M, F, Y, H, insertion, deletion,
Position 48 to
A, L, I, P, M, F, N, Y, D, H, insertion, deletion,
Position 51 to
V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 52 to
V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 53 to
A, V, L, I, W, M, F, N, Q, Y, S, D, E, H, deletion,
insertion,
Position 55 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H,
deletion, insertion,
Position 56 to
G, V, L, I, W, M, F, N, Q, Y, S, T, H,
Position 57 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, K, H,
Position 58 to
L, W, M, F, N, Y, R, insertion, deletion,
Position 61 to
V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to
G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 75 to
L,
Position 81 to
insertion,
Position 86 to
G, A, V, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
Position 87 to
A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 88 to
A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H,
Position 89 to
G, V, L, I, W, P, F, N, Y, T, E,
Position 91 to
G, A, V, L, I, W, P, M, N, Y, S, T, D, E, R, H, insertion,
deletion,
Position 92 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 94 to
G, V, L, I, W, P, M, F, N, Y, T, D, E, K, H, insertion,
deletion,
Position 96 to
L, W, F, Y, R, K, deletion,
Position 97 to
V, L, W, C, M, F, Y, H,
Position 98 to
deletion,
Position 101 to
V, I, W, M, F, N, Q, Y, H,
Position 102 to
V, L, I, W, M, F, Y, R, H, G, deletion,
Position 108 to
I, insertion,
Position 109 to
N, insertion,
Position 111 to
insertion,
Position 112 to
E, insertion,
Position 113 to
W, insertion,
Position 114 to
insertion,
Position 115 to
I, insertion,
Position 117 to
N, insertion,
Position 118 to
N, insertion,
Position 119 to
M, insertion,
Position 127 to
G, A, V, I, W, M, F, Y, R, H, L,
Position 133 to
A, L, I, W, M, F, Y, R,
Position 134 to
L, I, W, F, N, Q, Y, R, H, insertion, deletion,
Position 135 to
G, L, P, C, N, Q, T, R, H,
Position 136 to
G, A, W, P, N, Y, S, T, D, E, H, insertion, deletion,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 138 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 139 to
G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 141 to
G, V, L, I, W, P, M, F, Q, S, D, E, H, insertion, deletion,
Position 142 to
G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 144 to
L, W, P, M, F, N, Q, Y, S, D, E, R, H, insertion, deletion,
Position 145 to
G, V, L, I, W, M, F, Q, Y, D, E, R, H, insertion, deletion,
Position 146 to
G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 147 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion,
deletion,
Position 148 to
G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 156 to
V, I, W, F, R,
Position 158 to
V, L, I, W, M, F, Y,
Position 160 to
W, M, F, Y, R, H,
Position 161 to
I, W, M, F, Y, H,
Position 162 to
I, W, F, Y, R,
Position 163 to
V, W, M, F, H,
Position 167 to
R, K,
Position 169 to
C, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y,
Position 170 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 171 to
D, deletion,
Position 174 to
G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 176 to
G, A, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 182 to
A, V, L, I, W, C, M, F, N, Q, Y, S, T, D, E, H, deletion,
Position 186 to
G, A, V, L, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 188 to
G, A, V, L, W, F, S, R, K, deletion,
Position 191 to
A, V, L, I, W, M, F, Y, T, R, H, deletion,
Position 192 to
G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 193 to
G, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 195 to
W, P, Y, deletion,
Position 196 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, H,
insertion, deletion,
Position 197 to
G, V, L, I, W, P, M, F, Q, Y, S, T, H, insertion, deletion,
Position 198 to
G, A, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 203 to
V, F, Y, R, H,
Position 205 to
W, F, Y, R, K,
Position 215 to
A, L, I, W, M, F, Y,
Position 216 to
A, L, I, W, M, F, Y, R,
Position 217 to
W, R,
Position 219 to
G, A, V, L, I, W, F, Y, R, H,
Position 233 to
insertion,
Position 234 to
I, insertion,
Position 236 to
insertion,
Position 237 to
insertion,
Position 238 to
insertion,
Position 239 to
insertion,
Position 240 to
insertion,
Position 243 to
insertion,
Position 246 to
insertion,
Position 247 to
G, V, I, W, P, F, Y, S, T, R, insertion, deletion,
Position 249 to
L, W, P, F, S, D, E, H, insertion, deletion,
Position 252 to
G, A, W, P, N, Q, Y, T, E, R, H, insertion, deletion,
Position 254 to
G, V, L, I, W, M, F, N, Q, Y, S, D, E, R, H, insertion,
deletion,
Position 262 to
G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H, insertion,
deletion,
Position 264a to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
Position 270 to
G, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H, insertion,
deletion,
Position 273 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 274 to
W, P, M, F, N, Q, Y, T, D, E, R, H,
Position 275 to
G, A, V, L, I, W, M, F, N, Y, T, D, insertion, deletion,
Position 276 to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
with the proviso that the amino acids of the parent enzyme are substituted
to another amino acid.
152 . The protein variant of claim 135 having modified immunogenicity as compared to its parent protein having at least 81% homology to SEQ ID NO: 33 comprising one or more of the following substitutions corresponding to any of the following in SEQ ID NO: 33:
Position 5 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 22 to
G, V, L, I, W, M, F, Y, S, T, insertion, deletion,
Position 26 to
G, A, V, L, I, W, M, F, N, Q, Y, S, T, D, E, R, H, insertion,
deletion,
Position 28 to
G, A, V, L, I, W, P, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 37 to
L, I, W, M, F, N, Q, Y, S, R, H,
Position 40 to
V, L, I, W, M, F, N, Q, Y, T, R, H,
Position 44 to
G, V, L, I, W, P, M, F, Y, S, T,
Position 51 to
V, L, I, W, M, F, N, Y, R, deletion, insertion,
Position 52 to
V, L, I, W, M, F, Y, S, T, R, deletion, insertion,
Position 55 to
G, A, V, L, I, W, C, M, F, N, Q, Y, T, D, E, R, K, H,
deletion, insertion,
Position 58 to
L, W, M, F, N, Y, R, insertion, deletion,
Position 61 to
V, L, I, W, M, F, Y, insertion, deletion,
Position 64 to
G, V, L, I, W, P, C, M, F, N, Q, Y, S, T, D, E, R, K, H,
insertion, deletion,
Position 87 to
A, V, L, I, W, M, F, Q, Y, S, T, D, E, H,
Position 97 to
V, L, W, C, M, F, Y, H,
Position 98 to
deletion,
Position 101 to
V, I, W, M, F, N, Q, Y, H,
Position 102 to
V, L, I, W, M, F, Y, R, H, G, deletion,
Position 109 to
N, insertion,
Position 112 to
E, insertion,
Position 118 to
N, insertion,
Position 127 to
G, A, V, I, W, M, F, Y, R, H, L,
Position 137 to
G, A, V, I, W, P, M, N, Y, H, insertion, deletion,
Position 146 to
G, A, W, L, I, W, M, F, N, Q, Y, T, D, E, R, H,
insertion, deletion,
Position 156 to
V, I, W, F, R,
Position 158 to
V, L, I, W, M, F, Y,
Position 161 to
I, W, M, F, Y, H,
Position 188 to
G, A, V, L, W, F, S, R, K, deletion,
Position 192 to
G, L, I, W, M, N, Q, Y, S, T, D, R, H, deletion,
Position 194 to
W, N, Q, Y, D, H,
Position 195 to
W, P, Y, deletion,
Position 203 to
V, F, Y, R, H,
Position 216 to
A, L, I, W, M, F, Y, R,
Position 236 to
insertion,
Position 237 to
insertion,
Position 262 to
G, A, V, L, I, W, P, F, N, Q, Y, T, D, E, R, H,
insertion, deletion,
Position 264a to
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,
insertion, deletion,
with the proviso that the amino acids of the parent enzyme are substituted
to another amino acid.
153 . A subtilisin variant comprising one or more of the insertions, substitutions and/or deletions in any of the positions of claim 136 .
154 . The variant of claim 153 , wherein the subtilisin has at least 80% homology to SEQ ID NO; 10.
155 . A composition comprising the protein variant of claim 135 .
156 . A DNA construct comprising a DNA sequence encoding a protein variant of claim 135 .
157 . An expression vector comprising a DNA construct of claim 156 .
158 . A host cell which is capable of expressing a polypeptide and comprising a DNA construct of claim 156 .
159 . A host cell which is capable of expressing a polypeptide and which is transformed by an expression vector of claim 157 .
160 . The host cell of claim 159 , which is a fungal cell, an insect cell, a mammalian cell, or a plant cell.
161 . A method of producing a protein variant having reduced immunogenicity as compared to the parent protein, comprising:
(a) culturing the host of claim 159 in a suitable culture medium to obtain expression and secretion of the protein into the medium, followed by (b) isolation of the protein from the culture medium.Cited by (0)
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