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Artificial protein scaffolds

A protein and fusion protein technology, applied in the field of artificial protein scaffolds, can solve problems such as interference with scaffold function

Inactive Publication Date: 2011-04-13
MERCK PATENT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the use of natural proteins as scaffolds always has an inherent risk that the unknown biological properties of the natural protein will interfere with its function as a scaffold in a given situation

Method used

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  • Artificial protein scaffolds
  • Artificial protein scaffolds
  • Artificial protein scaffolds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1. Thermodynamic properties of scaffolds with peptide insertions

[0141] To confirm that RD1.3 is suitable as a scaffold containing large random peptide loops, loops 12, 34 and 56 were each replaced with eight glycines. These were chosen because glycine is the most damaging of all amino acids from a backbone entropy standpoint - if the protein has 8 glycines and is still folded and stable, then most other reasonably soluble random sequences The protein should also fold. Another sequence - a 15 amino acid loop "S-peptide" - was also inserted into the RD1.3 protein, alone and in combination with a glycine loop. The S-peptide is the part of the RNase-S enzyme that is known to bind to and complete the truncated enzyme, thereby restoring function. With this peptide as a loop insert, binding and enzymatic assays can be provided to demonstrate the ability of RD1.3 to display useful loops. Figure 8 The respective amino acid sequences of these test proteins are sh...

Embodiment 2

[0143] Example 2. Designed scaffold

[0144] A variety of proteins related to Top7 were designed to be used as protein scaffolds. The amino acid sequence of the protein is Figure 9 Alignments shown are described. as in Figure 9 As evident in , insertions in each of loops 12, 23, 34, 45, 56 and 67 were successfully engineered with or without point mutations at multiple positions throughout the scaffold. These proteins are expected to be at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical to one or more of these proteins or to the alpha-helices and beta-strands of one or more of these proteins Other related proteins that are identical, at least 90% identical, or at least 95% identical can be used as scaffolds and as the basis for protein libraries incorporating one or more heterologous sequences (as described in this application).

Embodiment 3

[0145] Example 3. Design and Synthesis of Exemplary Library RD1Lib1

[0146] To construct a library of genes with variable peptide loops, the following techniques were employed. First, a set of amino acids and a frequency distribution are selected as shown in the table below.

[0147] amino acid

percentage

Tyr

25

Ser

17

Leu

10

Ala

10

[0148] Asn

10

Gly

5

Ile

5

Asp

5

Arg

5

Pro

5

Trp

3

[0149] In this particular library construction, only 11 amino acids were selected. It will be apparent to those skilled in the art of protein engineering that various amino acids and distributions can be used. It is often useful to avoid the use of cysteine ​​and selenocysteine, since cysteine ​​can lead to undesired disulfide bond formation, and selenocysteine ​​is blocked by UGA codons (which can also be interpreted as codes for...

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Abstract

The present invention provides proteins having one or more similarities to the artificial protein Top7 or to a Top7 derivative. Proteins of the invention have one or more loops that are longer than the corresponding loops of Top7, and / or that bind to a preselected target molecule. The invention also provides nucleic acids and cells useful in producing the proteins and methods for their use.

Description

field of invention [0001] The present invention generally relates to artificial protein scaffolds and their design, production and use. In particular, the present invention relates to artificial protein scaffolds derived from the TOP 7 fold of the protein. Background technique [0002] Nature has provided many proteins in which short peptides of various sequences can be embedded. Antibodies are a well-known example and have an antigen-binding domain defined by heavy and light chain variable regions, where each variable region includes complementarity determining regions (CDRs) interposed between framework regions (FR). The CDR3 loops of both antibody heavy and light chains are formed by a process in which exonucleases and terminal transferases operate to insert essentially random DNA sequences encoding peptide loops into each V gene. When this process is combined with the more restricted diversity present in the CDR1 and CDR2 loops, VH and VL domains pair randomly, resulti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/62G06F17/50
CPCC07K14/00C12N15/1093
Inventor J·H·戴维斯
Owner MERCK PATENT GMBH
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