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Hemopexin-like structure as polypeptide-scaffold

A target molecule and specific technology, which is used in the field of preparing polypeptides with specific binding properties to predetermined target molecules

Inactive Publication Date: 2008-01-02
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, as already mentioned above, the bottleneck of protein knockout technology is that not all antibodies expressed intracellularly as affinity molecules are functionally produced ("The Disulfide Bond Problem", Visintin, M. et al., J. Immun. Meth. 290 (2004) 135-153)

Method used

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  • Hemopexin-like structure as polypeptide-scaffold
  • Hemopexin-like structure as polypeptide-scaffold

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1278] Example 1 Overlap extension ligation PCR (OEL-PCR)

[1279] Linear expression elements are assembled in a modular fashion by a two-step PCR protocol utilizing the principle of overlapping DNA ligation. In standard PWO-PCR, the intronless open reading frame is amplified with sequence-specific end-bridging primers, generating homologous sequences that overlap the flanking DNA sequences. 2 μl of the first PCR mix containing approximately 50 ng of the extended gene fragment (gene module) was transferred to the second PWO-PCR mix. To this mixture was provided 50ng to 100ng of pre-generated DNA fragments (promoter module and terminator module) and the respective sequence-specific end primers at 1 μM each. Typically, the second PCR step contains 30 cycles. The physical parameters of the PCR properties are adjusted according to the needs of the DNA fragments to be ligated.

Embodiment 2

[1280] Embodiment 2 synthetic PEX2 DNA library

[1281] Amino acids 64, 65, 86, 112, 113, 114, 130, and 132 encoding the hemopexin-like domain (equivalent to 528 (Gln), 529 (Glu), 550 (Arg ), 576(Lys), 577(Asn), 578(Lys), 594(VaI) and 596(Lys)) of the PEX2 triplet codons were randomized by the NNK-motif. The human wild-type PEX2 DNA sequence is divided into three sequence parts. Standard PWO-PCR to which 10 ng of vector template pIVEX2.1MCS PEX2 and 1 μM each of primers PEX2forw (SEQ ID NO: 63) and PEXR4 (SEQ ID NO: 64) were provided amplified a 1 bp-218 bp fragment. The 402bp-605bp fragment was amplified in standard PWO-PCR with 10 ng vector template pIVEX2.1MCS PEX2 and 1 μM each of primers PEXF4 (SEQ ID NO: 65) and PEX2rev (SEQ ID NO: 66). The formed sequence 196bp-432bp overlaps with the DNA fragment 1bp-218bp and 402bp-605bp and is primer PEXF1 (SEQ ID NO: 67) and PEXR1 (SEQ ID NO: 68) each 1 μ M and PEXR3 (SEQ ID NO: 69), PEXR2 ( SEQ ID NO: 70) and PEXF2 (SEQ ID NO: 7...

Embodiment 3

[1282] Example 3 Cell-free protein transcription and translation in vitro

[1283] Linear expression elements were transcribed and translated in the RTS 100HY E. coli system according to the manufacturer's instructions. Linear DNA templates (100ng-500ng) were incubated at 30°C. Optionally 6 μl of GroE supplement (Roche) was added.

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Abstract

The invention concerns a method for the generation of a polypeptide with specific binding properties to a predetermined target molecule which are not naturally inherent to that polypeptide. At the same time an optimization of the binding specifity and a process of production are described. The invention further concerns a method for the identification and modification of specific amino acid positions within a polypeptide scaffold.

Description

[0001] The present invention relates to methods for preparing a polypeptide having specific binding properties for a predetermined target molecule, wherein said specific binding properties are not naturally inherent to the polypeptide. Also the invention describes methods of optimizing binding specificities and methods of producing them. The present invention also relates to methods for identifying and modifying alterable amino acid positions in polypeptide scaffolds. technical background [0002] The number of patent applications and publications related to affinity reagents has steadily increased in recent years. Most of them involve antibodies, ie monoclonal or polyclonal immunoglobulins. Only a small part deals with possible alternatives. One of these alternatives is the use of protein scaffolds. This concept requires a stable protein architecture that tolerates multiple substitutions or insertions at the level of primary structure (Nygren, P-A., Skerra, A., J. Immun. M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N9/64C12N15/10C12N15/63G01N33/68
CPCC12N15/1034G01N33/6803C12N15/63C12N9/6491C07K14/47
Inventor M·兰岑多费尔M·施拉伊姆尔
Owner F HOFFMANN LA ROCHE & CO AG
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