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Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains

A technology of lectin-like and scaffold structure is applied in the field of protein combinatorial library with scaffold structure of C-type lectin-like domain, which can solve the problem of increasing the number of CTLDs and the like

Inactive Publication Date: 2004-03-24
ANAPHORE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In physiological functional units, the number of CTLDs can often be increased by assembling single-copy CTLDs into larger structures

Method used

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  • Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains
  • Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains
  • Combinatorial libraries of proteins having the scaffold structure of c-type lectin-like domains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0413] Construction of Escherichia coli expression plasmids and phagemids derived from tetrapodrin

[0414] Take advantage of QuickChange TM Site-directed mutagenesis kit (Stratagene, La Jolla, CA) and performed as described in the manual, starting with the expression plasmid pT7H6-rTN 123 [Holtet et al. (1997)], 4 consecutive series of site-directed mutagenesis experiments were performed, An expression plasmid pT7H6FX-htlec encoding the FX-htlec (SEQ ID NO: 01 ) portion of the full-length H6FX-htlec fusion protein was constructed. Mismatched primer pairs to introduce the desired mutations were supplied by DNA Technology (Aarhus, Denmark). Figure 5 shows a schematic diagram of the obtained pT7H6FX-htlec expression plasmid, and SEQ ID NO: 01 gives the nucleotide sequence encoding the insert segment of FX-htlec. Figure 6 shows the amino acid sequence of the FX-htlec portion of the H6FX-htlec fusion protein and is given in SEQ ID NO:02.

[0415]By amplifying and subcloning int...

Embodiment 2

[0422] Confirmation of successful display of Phtlec and PhTN3 on phage

[0423]In order to verify that the fusion protein of Phtlec and PhTN3 Gene III can indeed be displayed using recombinant phage particles, the phagemids pPhtlec and pPhTN3 (described in Example 1) were transformed into Escherichia coli TG1 cells and infected with helper phage M13KO7 to produce Recombinant phages were obtained. Recombinant phages were separated by precipitation with polyethylene glycol (PEG8000), and ELISA-type "sandwich" tests were performed on Phtlec and PhTN3 phage preparations and helper phage samples. Protein and bovine serum albumin (BSA) and blocked in skim milk or skim milk / EDTA. Briefly, pPhtlec and pPhTN3 phagemid-transformed TG1 cell cultures were grown at 37°C in 2×TY-medium supplemented with 2% glucose and 100 mg / L ampicillin until their A 600 reach 0.5. At that time, helper phage M13KO7 was added to it to a concentration of 5 × 10 9 pfu / mL. Cultures were incubated for an a...

Embodiment 3

[0427] Confirmation of the authentic ligand-binding properties of Phtlec and PhTN3 displayed on phage

[0428] The apogroup of the CTLD domain of human tetramantin binds specifically to the human plasminogen kringle 4 domain in a lysine-sensitive manner [Graversen et al. (1998)]. Tetramantin binding to plasminogen is inhibited by calcium or lysine analogs such as AMCHA (6-amino-cyclohexanoic acid), where calcium binds to two of the ligand-binding sites of the CTLD domain point (Kd is about 0.2mM), while lysine analogs can specifically bind to two stronger lysine binding sites in plasminogen (Kd is about 15mM), these two sites One is positioned in kringle 1 and the other is positioned in kringle 4.

[0429] To demonstrate the specific AMCHA-sensitive binding of human Phtlec and PhTN3 phage to human plasminogen, an ELSISA assay similar to that applied to demonstrate the presence of Phlec and PhCTLD GIII fusion proteins displayed on phage particles (see Example 2) was designed. ...

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Abstract

A novel family of protein libraries comprising CTLDs (C-type Lectin-Like Domains) in which internal polypeptide loop-regions lining the ligand binding sites in CTLDs have been replaced with ensembles of completely or partially randomised polypeptide segments. Tetranectin CTLDs were chosen as framework for the preferred embodiment of the invention; and versatile phagemid vectors useful in the generation and manipulation of human and murine tetranectin CTLD libraries are disclosed as part of this invention. Tetranectin CTLDs in monomeric as well as in trimeric form are efficiently displayed as gene III fusions in fully functional form by the recombinant fd phage display vector. CTLD derivatives with affinity for new ligands may readily be isolated from libraries of vectors displaying CTLDs, in which loop-regions have been randomised, using one or more rounds of enrichment by screening or selection followed by amplification of the enriched subpopulation in each round. The efficiency with which protein products containing CTLDs with new binding properties can be produced, e.g. by bacterial expression and in vitro refolding, in mono-, tri-, or multimeric formats provides important advantages in terms of simplicity, cost and efficiency of generation, production and diagnostic or therapeutic applications in comparison to recombinant antibody derivatives.

Description

field of invention [0001] The present invention describes a system related to the generation of randomized libraries of ligand-binding protein units derived from proteins containing the so-called C-type lectin like domain (CTLD), and wherein the C The carbohydrate recognition domain (CRD) of lectins represents an example of this family of protein domains. Background of the invention [0002] C-type lectin-like domains (CTLDs) are a family of protein domains identified in proteins isolated from various animals (for review see Drickamer and Taylor (1993) and Drickamer (1999)). Initially, the CTLD domain was identified as a ubiquitous domain of so-called C-type lectins (calcium-dependent carbohydrate-binding proteins) and named the "carbohydrate recognition domain" ("CRD"). Recently, it was found that this domain is shared by various eukaryotic proteins, some of which do not bind sugar moieties, so this canonical domain was named CTLD. [0003] CTLD has been reported to bind ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/47C07K19/00C12N15/10C12N15/12C12P21/02
CPCC40B40/08C40B40/02C40B50/06C07K14/4726C12N15/1044C07K14/47
Inventor M·伊泽罗德特T·L·霍尔泰特N·J·H·格拉沃森H·C·瑟格森
Owner ANAPHORE INC
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