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Purified and isolated protein zero related (PZR) and therapeutic and screening methods using same

a technology of purified and isolated proteins and screening methods, applied in the field of purified proteins, can solve problems such as serious deficiency in the art of lack of knowledg

Inactive Publication Date: 2006-01-19
VANDERBILT UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This lack of knowledge represents a serious deficiency in the art in view of the effects of SHP-2 as described above.

Method used

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  • Purified and isolated protein zero related (PZR) and therapeutic and screening methods using same
  • Purified and isolated protein zero related (PZR) and therapeutic and screening methods using same
  • Purified and isolated protein zero related (PZR) and therapeutic and screening methods using same

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example 1

Isolation and Purification of PZR

[0256] Purification of PZR from 293 cells overexpressing the catalytically inactive mutant of SHP-2. The stably transfected 293 cells overexpressing the catalytic inactive mutant of SHP-2 were grown in DMEM / High containing 10% calf serum and 100 unit / ml each of penicillin and streptomycin and 0.25 mg / ml G418 sulfate. After growing to confluency, the cells were treated with 0.1 mM pervanadate for 20 min before harvesting in ice-cold phosphate-buffered saline. The collected cells were broken up with a Dounce glass homogenizer in Buffer A containing 25 mM β-glycerophosphate (pH 7.3), 10 mM EDTA, 2 mM EDTA, 0.2 mM Na3VO4, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 2 μg / ml leupeptin, 1 μM pepstatin A, and 1 μg / ml aprotinin. Nuclear pellets were removed by centrifugation at 800×g for 20 min, and the remaining postnuclear extract was further centrifuged at 100,000×g for 45 min to give a clear cytosolic supernatant and a pelleted membrane fract...

example 2

Evaluation of the Association of PZR with SHP-2 In Vivo

[0266] Identification and purification of a 43 kDa hyperphosphorylated protein. All PTPs contain a highly conserved cysteinyl residue within their catalytic centers, which is directly involved in the formation of a thiophosphate intermediate essential for the catalysis (reviewed in Fischer, E. H., et al. (1991) Science 253:401-6 and in Walton, K. M. and Dixon, J. E. (1993) Annu. Rev. Biochem. 62:101-20). Mutation of this cysteinyl residue to serine impairs the phosphatase activity. The Cys-to-Ser mutants of SHP-1 and SHP-2 display dominant negative effects and cause hyperphosphorylation of specific cellular proteins on tyrosine as previously described (Zhao, Z., et al. (1995) J. Biol. Chem. 270:11765-17769; Su, L., et al. (1996) J. Biol. Chem. 271:10385-10390. ). In human embryonic kidney 293 cells, expression of the catalytically inactive Cys-to-Ser mutant form of SHP-2 resulted in tyrosine phosphorylation of 43 and 95 kDa pro...

example 3

Expression of a Soluble PZR Polypeptide

[0286] The expression of a soluble form of PZR was performed in 293 cells. The cDNA encoding the signal sequence and the extracellular portion of PZR was cloned into the pCDNA3 vector, and the DNA construct was used to transfect 293 cells. The culture medium was analyzed for expression of soluble PZR by using Western blotting with an anti-PZR antibody. Three forms of soluble PZR representing different degrees of glycosylation were seen at 14.4, 21.5 and 29 kDa, respectively. Representative amino acid and nucleic acid sequences for soluble PZR are set forth in SEQ ID NOs:33-40.

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Abstract

Isolated and purified proteins and nucleic acids including a novel member of the immunoglobulin super-family characterized as having SHP-2 binding activity and cell signaling activity and called protein zero related or PZR, and cDNA encoding the same. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also disclosed, along with methods of producing each. Isolated and purified antibodies to PZR, and methods of producing the same, are also disclosed. PZR is characterized as having SHP-2 binding activity and cell signaling activity and thus, therapeutic methods involving these activities are also disclosed.

Description

PRIORITY APPLICATION INFORMATION [0001] This application is a regular United States patent application under 35 U.S.C. § 111(a) based on and claiming priority to U.S. Provisional Application Ser. No. 60 / 106,459 filed Oct. 30, 1998, the entire contents of which are herein incorporated by reference.GRANT STATEMENT [0002] This work was supported by National Institutes of Health (NIH) grants HL57393, CA75218 and CA-69485. Thus, the U.S. Government has certain rights in the invention.TECHNICAL FIELD [0003] The present invention relates generally to isolated and purified proteins that modulate SHP-2 biological activity and modulate cell signaling, and to nucleic acids encoding the same. More particularly, the present invention relates to an isolated and purified transmembrane protein designated as “protein zero related” or “PZR” that binds the tyrosine phosphatase SHP-2, and an isolated and purified polynucleic acid encoding the same. Table of AbbreviationsBSABovine serum albuminEGFepide...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/705C12N5/06C07K16/44
CPCC07K16/2803C07K14/70503
Inventor ZHAO, ZHIZHUANG
Owner VANDERBILT UNIV
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