Molecules and chimeric molecules thereof

A technology of chimeric molecules and proteins, applied in growth factors/inducing factors, drug combinations, animal/human proteins, etc., can solve problems such as pollution, inapplicability to clinical applications, and harmful transplantation applications

Inactive Publication Date: 2008-06-11
APOLLO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, stem cells are routinely maintained in media that include non-human proteins, which is not suitable for clinical use due to the possibility of contamination with non-human infectious substances
Further, stem cell culture in non-human derived media may result in the introduction of non-human carbohydrate moieties thus compromising transplantation applications

Method used

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  • Molecules and chimeric molecules thereof
  • Molecules and chimeric molecules thereof
  • Molecules and chimeric molecules thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1055] Cloning of proteins of the invention

[1056] (a) Preparation of pIRESbleo3-Fc construct

[1057] The DNA sequence encoding the Fc domain of human IgG1 was amplified by polymerase chain reaction (PCR) from an EST cDNA library (Clone ID 6277773, Invitrogen) using cDNAs incorporating restriction enzymes BamH1 and BstX1 sites, respectively. Forward primer (SEQ ID NO: 21) and reverse primer (SEQ ID NO: 22). This amplicon was cloned into the corresponding restriction site in pIRESbleo3 (Cat. No. 6989-1, BD Biosciences) to make construct pIRESbleo3-Fc. Digestion of pIRESbleo3-Fc with BamH1 and BstX1 released an insert of the expected size of 780 bp as determined by gel electrophoresis.

[1058] (b) Preparation of DNA constructs expressing protein or protein-Fc

[1059] The DNA sequence encoding the protein or its extracellular domain was amplified by PCR from the EST cDNA library using forward and reverse primers with restriction enzyme sites introduced according to Table ...

Embodiment 2

[1067] (a) Preparation and purification of EPO of the present invention

[1068] (i) Preparation of EPO of the present invention

[1069] On day 0, use 3 × 10 cells of the transformed embryonic human kidney cell line 7 Cell seeding five 500cm 2 Tissue culture dishes (Corning) with cells such as HEK 293, HEK 293c18, HEK 293T, 293CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene) 293A (Invitrogen). Cells were inoculated into 90ml of Dulbecco's modified Eagle's medium / Ham's nutrient mixture F12 (DMEM / F12) (JRH Biosciences) per plate, and the medium was supplemented with 10% (v / v) heat-inactivated fetal calf serum (FCS , JRH Biosciences), 10 mM HEPES (Sigma), 4 mM L-glutamine (Ameresco) and 1% (v / v) penicillin-streptomycin (JRH Biosciences).

[1070] On the first day, transfection was performed with calcium phosphate. Before transfection, the medium in each plate was replaced with 120 ml of fresh DMEM / F12. Prepare a calcium phosphate / DNA precipitate by adding 1200 μg of...

Embodiment 3

[1145] (a) Features of EPO of the present invention

[1146] (i) Two-dimensional polyacrylamide electrophoresis

[1147] Samples collected from Example 2(a) were buffer exchanged through a dialysis or desalting column (Pharmacia HR 10 / 10 Fast Desalting Column) into repurified (18 MOhm) water and dried with a SpeedVac concentrator. Then, the sample was redissolved in 240 μl MSD buffer (5M urea, 2M thiourea, 65mM DTT, 2% (w / v) CHAPS, 2% (w / v) thiobetaine 3-10, 0.2% (v / v) vehicle ampholyte, 40 mM Tris, 0.002% (w / v) bromophenol blue, water) and centrifuged at 15000 g for 8 minutes.

[1148] Isoelectric focusing (IEF) was performed with precast 11 cm or precast 17 cm gels pH 3-10 immobilized pH gradient IEF strips (BioRad). IEF strips were rehydrated with samples in closed tubes at room temperature for at least 6 hours. The IEF strips were placed in the focusing chamber and covered with liquid paraffin. IEF was performed at 100V for 1 hour, 200V for 1 hour, 600V for 2 hours, 1...

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Abstract

The present invention relates generally to the fields of proteins, diagnostics, therapeutics and nutrition. More particularly, the present invention provides an isolated protein molecule such as EPO, Flt3-Ligand, Flt3, PDGF-B or VEGF-165 or chimeric molecules thereof comprising at least a portion of the protein molecule, wherein the protein or chimeric molecule has a profile of measurable physiochemical parameters which is indicative of, associated with or forms the basis of, one or more pharmacological traits. The present invention further contemplates the use of the isolated protein or chimeric molecule thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and / or research applications.

Description

technical field [0001] The present invention generally relates to proteology, diagnostics, therapeutics and nutrition. In particular, the invention provides isolated protein molecules, such as EPO, Flt3-ligand, Flt3, PDGF-B or VEGF-165 or chimeric molecules comprising at least a part of said protein molecules, wherein the protein or chimeric molecules have the A characteristic of a measured biochemical parameter that represents, correlates or forms the basis of one or more pharmacological properties. The present invention further contemplates the use of the isolated protein or chimeric molecule thereof within the context of diagnostic, prophylactic, therapeutic, nutritional and / or research applications. Background technique [0002] Any reference to prior art in this specification is not and should not be construed as an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge. [0003] Growth factors and their receptors are im...

Claims

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Application Information

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IPC IPC(8): C07K14/505A61P35/00C07H21/04
Inventor J·D·普里斯特A·D·沃茨J·S·惠特克T·A·多马加拉R·J·辛普森G·R·皮尔金顿C·A·利德尔I·贝姆C·M·Y·李
Owner APOLLO LIFE SCI
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