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Novel genes related to glutaminyl cyclase

An amino acid, similarity technology, applied in genetic engineering, plant genetic improvement, transferase, etc., can solve problems such as incurable multiple sclerosis

Active Publication Date: 2009-11-04
VIVORYON THERAPEUTICS NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0080] There is currently no known cure for multiple sclerosis

Method used

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  • Novel genes related to glutaminyl cyclase
  • Novel genes related to glutaminyl cyclase
  • Novel genes related to glutaminyl cyclase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0333] Embodiment 1: the preparation of people's isoQC

[0334] Cell Lines and Media

[0335] African green monkey kidney cell line COS-7, human neuroblastoma cell line SH-SY5Y, human astrocytoma cell line LN405, human keratinoma cell line HaCaT, and human hepatocellular carcinoma cell line Hep-G2 in suitable Cell culture medium (DMEM, 10% FBS for COS-7, SH-SY5Y, LN405, HaCaT), (RPMI1640, 10% FBS for Hep-G2), in 5% CO 2 (HaCaT, Hep-G2, COS-7) or 10% CO 2 (SH-SY5Y, LN405) were cultured at 37°C in a humidified atmosphere.

[0336] Analysis of human isoQC expression using RT-PCR

[0337] Total RNA was isolated from SH-SY5Y, LN405, HaCaT and Hep-G2 cells using RNeasy mini kit (Qiagen) and reverse transcribed by SuperScript II (Invitrogen). Subsequently, the resulting cDNA was reacted in a 25 μl reaction with Herculase Enhanced DNA-polymerase (Stratagene) using primers isoQCh-1 (sense, SEQ ID NO: 53) and isoQCh-2 (antisense, SEQ ID NO: 54) A 1:12.5 dilution of the product ...

Embodiment 2

[0343] Example 2: Preparation and expression of isoQC in mammalian cell culture

[0344] Molecular Cloning of Plasmid Vector Encoding Human isoQC-EGFP Fusion Protein

[0345] All cloning methods were performed using standard molecular biology techniques. For expression of human isoQC-EGFP fusion protein in human cells, the vector pEGFP-N3 (Invitrogen) was used. The cDNA of native human isoQC starting at methionine I or at methionine II was fused amino-terminally in-frame to a plasmid encoding enhanced green fluorescent protein (EGFP). Primers isoQC EGFP-1 Met I (SEQ ID NO: 57) and isoQC EGFP-3 (SEQ ID NO: 59) were used to amplify human isoQC starting from methionine I and primers isoQC EGFP-2 Met II (SEQ ID NO :58) and isoQC EGFP-3 (SEQ ID NO:59) were used to amplify human isoQC starting at methionine II. Using the restriction sites EcoRI and SalI, the fragment was inserted into vector pEGFP-N3 (Invitrogen) and correct insertion was confirmed by sequencing. Subsequently,...

Embodiment 3

[0352] Example 3: Immunohistochemical staining of human isoQC in mammalian cells

[0353] Transfection and histochemical staining of COS-7 and LN405

[0354] To express the human isoQC-EGFP fusion protein starting with methionine I or methionine II, COS-7 and LN405 were cultured in 6-well dishes containing coverslips. Cells were grown to 80% confluency, transfected using Lipofectamin2000 (Invitrogen) according to manufacturer's manual and incubated in transfection solution for 5 hours. Thereafter, the solution was replaced with the appropriate growth medium and the cells were incubated overnight.

[0355] The next day, cells were washed twice with D-PBS (Invitrogen) and fixed with ice-cold methanol at -20°C for 10 minutes, followed by 3 washing steps with D-PBS for 10 minutes at room temperature. For Golgi band staining, COS-7 and LN405 were incubated with rabbit anti-mannosidase II polyclonal antibody (Chemicon) at a 1 :50 antibody dilution in D-PBS for 3 hours. To stain...

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Abstract

The present invention relates to novel glutaminyl-peptide cyclotransferase-like proteins (QPCTLs), which are isoenzymes of glutaminyl cyclase (QC, EC 2.3.2.5), and to isolated nucleic acids coding for these isoenzymes, all of which are useful for the discovery of new therapeutic agents, for measuring cyclase activity, and for determining the inhibitory activity of compounds against these glutaminyl cyclase isoenzymes.

Description

field of invention [0001] The present invention relates to novel glutamine peptide cyclization transferase-like protein (QPCTL) as the isozyme of glutamyl cyclase (QC, EC 2.3.2.5), and to the isolated nucleic acid of these isozymes of encoding, described The isolated nucleic acids from are useful for the discovery of new therapeutic substances, for measuring cyclase activity and for determining the inhibitory activity of compounds against these glutamyl cyclase isozymes. Background of the invention [0002] Glutamyl cyclase (QC, EC 2.3.2.5) catalyzes the conversion of amino-terminal glutamine residues to pyroglutamic acid (pGlu * ) is intramolecularly cyclized, thereby releasing ammonia. QC was first isolated from the emulsion of the tropical plant Carica papaya by Messer in 1963 (Messer, M.1963 Nature 4874, 1299). Corresponding enzymatic activity was found in animal pituitary after twenty-four years (Busby, W.H.J. et al. 1987 JBiol Chem 262, 8532-8536; Fischer, W.H. and S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10A61K31/4164C07D233/54C07D235/06C07D403/12C07D405/12C07D417/12
Inventor S·席林H·钦尼斯J-U·拉赫菲尔德H-U·穆德特J－U·拉赫菲尔德H－U·穆德特
Owner VIVORYON THERAPEUTICS NV
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