Scaffold proteins

A technology of heterologous peptides and amino acids, applied in the field of scaffold proteins, can solve problems such as no regulation or change of thermal stability

Pending Publication Date: 2020-05-08
AVACTA LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There is no teaching in this document on how to adjust or change the thermal stability

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1339] Example 1 - Mutations can be independently combined to modulate stability

[1340] The following mutations were made relative to SEQ ID NO: 1:

[1341]

[1342] These all increased the Tm of hSteA Y35W.

[1343] This suggests that the order of mutations does not matter. In other words, this demonstrates that stability (eg, Tm) modulating mutations taught herein can be independently made or independently combined.

[1344] An advantage of the present invention is that the specific stability-modulating mutations taught herein are independent of each other to achieve their respective effects.

Embodiment 2

[1345] Example 2: Heterologous Peptide Inserts

[1346] Testing the heterologous peptide GGSGGSGGS inserted into L2 and L4

[1347] 3 different positions per ring (9 combinations)

[1348] Measuring Thermal Stability on Optim 2

[1349] 3r2 measured without denaturant but stability out of range

[1350] 3r2 was measured again in the presence of 1M GuHCl to bring the Tm in range

[1351] 3t4 was measured in the presence of SYPRO Orange since there is no intrinsic tryptophan fluorescence in this peptide

[1352] In summary, the options for various examples are shown below:

[1353] SQT (SEQ ID NO: 24 of WO 2009 / 136182)

[1354]

[1355] 3r2 (SEQ ID NO: 19)

[1356]

[1357] 3t4 (SEQ ID NO: 23)

[1358]

[1359] 3r2 (SEQ ID NO: 19) / t4 (SEQ ID NO: 23)

[1360]

[1361] 3r2 (SEQ ID NO: 19) / t4 (SEQ ID NO: 23)

[1362]

[1363] 3r2 (SEQ ID NO: 19) / t4 (SEQ ID NO: 23)

[1364]

[1365] The result is as figure 1 shown.

[1366] This indicates that insertio...

Embodiment 3

[1374] Example 3: Exemplary Scaffold Proteins

[1375] Exemplary scaffold proteins are shown.

[1376] In this experiment, Tm was measured by DSC (r / t) and Optim 2 (3r).

[1377] Exemplary holders for research applications:

[1378] 3r1-hSteA Y35W N32G V48D M65I Q42E T51L (A59VΔD61) (E29K K30E E33K) (SEQ ID NO: 18)

[1379] 3r2-hSteA Y35W N32G V48D M65I Q42E T51L (A59V G60NΔD61N62G) (E29K K30EE33K) (SEQ ID NO: 19)

[1380] Exemplary stents for therapeutic applications:

[1381] SEQ ID NO:20 3t1-hSteA N32G V48D

[1382] SEQ ID NO:21 3t2–hSteA N32G V48D M65I

[1383] SEQ ID NO:22 3t3–hSteA N32G V48D M65I T51L

[1384] SEQ ID NO:23 3t4–hSteA N32G V48D M65I Q42E

[1385] SEQ ID NO:24 3t5–hSteA N32G V48D M65I Q42E T51L

[1386] figure 2 Data are shown for Tm as a measure of thermal stability.

[1387] image 3 Tm measured at different pH values ​​are shown.

[1388] Figure 4 shows the CD spectrum.

[1389] We generated DSC data for the above exemplary scaffolds and ob...

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Abstract

The invention relates to a polypeptide, such as an Affimer polypeptide, comprising an amino acid sequence having at least 80% identity to amino acid residues 1 to 11, 13 to 15, 17 to 19, 21 to 25, 27to 28, 35 to 37, 39, 41, 43 to 44, 46 to 47, 49 to 50, 52 to 53, 55 to 58, 63 to 64, 66, 68 to 82, 84 to 85, and 87 to 98 of SEQ ID NO: 1; characterised in that said polypeptide comprises one or moremutations relative to SEQ ID NO: 1 selected from the group consisting of: T51L, T51V, M65V, N32G, A59I, L38A, V20I, A40I, L38V, A12I, A12V, I16L, V20L, Q26E, E29M, T31K, N32D, N32H, T34V, T34R, T34D,T34P, A40V, Q42D, T45I, T45V, V48E, V48G, V48A, T51F, T51A, A59L, L67I, (V20I, L38A), (V20L, L38A), (V20I, L38V), (V20L, L38V), (E29K, K30E, E33K), (Y54D, T83D, Q86E), (A59L, G60N, D61G, N62K), (A59V,D61N, N62K), (G60N, D61G, N62K), (G60N, delta D61, N62G), delta D61, (A59L, G60N, delta D61, N62G), (A59V, G60N, D61G, N62K), (A59I, G60N, D61G, N62K), (A59I, G60N, delta D61, N62G), (A59V, G60N, delta D61, N62G), (A59V, delta D61), (G60P, delta D61, N62P), (G60P, D61P, N62K), (G60P, delta D61, N62G), (G60P, D61G, N62K), (D61N, N62K) and (T83D, Q86E). The invention also relates to various methodsand nucleic acids.

Description

[0001] Background of the invention [0002] WO 2009 / 136182 discloses mutations of prior art scaffold STMs using D48L and G50S which are disclosed to result in increased expression in bacterial systems (page 22, lines 12-13). [0003] Both WO 2009 / 136182 and WO 2006 / 131749 have previously disclosed that mutations at the V48 site of wild-type cystatin A, particularly V48D, can be used to eliminate domain switching dimerization (WO 2006 / 131749, pp. 35, line 25). [0004] In WO 2009 / 136182 simultaneous mutations of E78A and L80R of the scaffold SQT have been disclosed, as well as simultaneous mutations of L82R and T83S in the SQM scaffold (seam-riding paragraph on pages 22 to 23 of WO 2009 / 136182). These simultaneous mutant pairs have been disclosed to exhibit high expression in E. coli. [0005] WO 2009 / 136182 discloses modified Stefin A scaffold proteins. In particular, the disclosure in this document focuses on the "position 4 mutation", which corresponds to the G4 site of cys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C12N15/62
CPCC07K14/8139C07K2319/00C07K16/2827C07K16/32C07K16/42C07K2318/20C07K2319/30C07K2319/31
Inventor 杰夫·威廉·普拉特格雷厄姆·罗伯特·派伊-史密斯·斯彭斯
Owner AVACTA LIFE SCI
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