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Crystalline TNF-alpha-converting enzyme and uses thereof

A technology for converting enzymes and crystals, applied in special data processing applications, enzymes, hydrolytic enzymes, etc., can solve problems such as organ failure and death

Inactive Publication Date: 2002-03-27
AMGEN INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can also eventually lead to organ failure and death

Method used

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  • Crystalline TNF-alpha-converting enzyme and uses thereof
  • Crystalline TNF-alpha-converting enzyme and uses thereof
  • Crystalline TNF-alpha-converting enzyme and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Expression, isolation and purification of embodiment 1-TACE polypeptide

[0094] A cDNA encoding the signal peptide, pro- and catalytic domains of TACE, amino acid residues 1-477, as disclosed by Black et al., "A Metalloproteinase disintigrin that releases tumor-necrosis factors from cells," Nature 385:729-733 (1997, February), whose Ser266 was changed to Ala, Asn452 was changed to Gln, and the sequence Gly-Ser-(His)6 was added to the C-terminus, was inserted into the expression vector of CHO cells. The TACE polypeptide is expressed in CHO cells and is secreted as a mixture of TACE polypeptides originating from Val212 or Arg215. The cells were cultured in the drug methotrexate, which kills cells that do not bind the carrier.

[0095] The expressed TACE polypeptide is then purified. Purification starts with 5 liters of medium containing the expressed TACE polypeptide. The medium is filled with a Millipore 10K cut-off, 1 ft 2 TFF's diafiltration unit concentrates to a...

Embodiment 2

[0104] Example 2 - Protein crystallization

[0105] A DNA construct containing the prodomain and catalytic domain (residues 1-477) of human TACE was fused to the sequence Gly-Ser-(His)6 to facilitate protein purification on a Ni-NTA affinity column. Chinese Hamster Ovary (CHO) cells were used for protein expression. The cells secrete a mixture of mature TACEs initiated with Val212 or Arg215. The TACE-containing fraction obtained from the Ni-NTA column contained octyl glucoside and the binding partner N-[D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanol)-L- 3-(tert-butyl)-glycyl-L-alanamine buffer for incubation. The final purification step is performed on a gel filtration column. Purified TACE was stored in a buffer (TACE buffer) containing 10 mM Tris / HCL pH 7.5, 100 mM NaCl, 10% glycerol and 1 mM inhibitor.

[0106] The concentration of TACE in the crystallization experiments was approximately 5 mg / ml, and TACE (in TACE buffer) and the following crystallization buffe...

Embodiment 3-X

[0114] Example 3 - X-ray Diffraction

[0115] Using the crystal described in Example 2, the first MAR300 imaging plate scanner was collected on a Rigaku-Denki rotating target copper anode X-ray generator (power is 5.4kW, producing graphite monochromatic CuKα rays) A set of data with a resolution of 2.5 Å. Diffraction data were processed with MOSFLM v.5.23 program and CCP4 package routines.

[0116] All attempts to solve the structure by molecular replacement using adamalysin II (an all-alanine model of adamalysin II) and other models failed to yield useful starting points for phase determination.

[0117] Therefore, determination of the positions of four independent zinc atoms was assisted by an anomalous difference Patterson synthesis. To determine MAD data, crystals were deep frozen in a nitrogen stream frozen to liquid nitrogen temperature. The crystals were first transferred to a freezing buffer containing 80% v / v Buffer D (0.1M sodium citrate pH5.4, 20% w / v PEG4000, 20...

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Abstract

A tumor necrosis factor-alpha converting enzyme (TACE) is produced, purified, and crystallized. The three-dimensional coordinates of the crystal are obtained by X-ray diffraction. The coordinates can be recorded on a computer readable medium, or are part of a video memory, where they can be used as part of a system for studying TACE. The coordinates are also used in designing, screening, and developing compounds that associate with TACE.

Description

[0001] Related application information [0002] This application claims priority from the following applications: U.S. Provisional Patent Application Serial No. 60 / 073,709 (filed February 4, 1998), U.S. Patent Application Serial No. 09 / 050,083 (filed March 30, 1998), which will be based on the 1999 Amendment to a US provisional application filed January 27), and a US provisional patent application entitled "Crystalline TNF-alpha-Converting Enzymes and Uses Thereof" (filed January 27, 1999). Background of the invention [0003] The cytokine tumor necrosis factor-α (TNFα) plays a role in inducing inflammatory responses and is known to be cytotoxic to tumor cells. However, when produced in excess, TNF[alpha] can also cause serious damage to the human body by ultimately leading to multi-organ failure and death. See Bemelmans et al., "Tumor Necrosis Factor: Function, Release and Clearance," Crit. Rev. Immun. 16: 1-11 (1996). [0004] Tumor necrosis factor-alpha is produced by act...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K1/14C12NC12N9/50C12N9/64G06F17/30
CPCC12N9/6489
Inventor R·A·布拉克R·J·帕克斯顿W·博德K·马斯科斯C·菲尔南德兹-卡塔兰J·M·陈J·I·莱文
Owner AMGEN INC
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