Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor

a biochemical precursor and isotopically-labeled technology, applied in the field of site-specific isotopically-labeled organic compounds, can solve the problems of reducing the yield of specific amino acids, and reducing the cost of production. the effect of cost minimization

Inactive Publication Date: 2006-12-28
FESIK STEPHEN +1
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach allows for cost-effective production of proteins and polypeptides with strong NMR signals, reducing interference and enhancing the accuracy of NMR drug discovery techniques.

Problems solved by technology

However, the application of this NMR drug discovery technique to target molecules isotopically enriched with 13C has been hampered by two drawbacks.
First, it is comparatively expensive to produce 13C-enriched target molecules in any useful quantities.
For example, the production of proteins by genetically modified microorganisms grown in nutrient media containing commercially available uniformly-labeled glucose (glucose-13C6) is expensive.
Alternatively, the production of 13C-labeled proteins by including uniformly 12C-labeled amino acids in the nutrient medium is similarly expensive.
Second, the biomolecules produced using glucose-13C6 or commercially available uniformly 13C-enriched amino acids are not ideally suited for the NMR correlation spectra technique.
Since the NMR technique depends upon detection of spatial spin coupling (i.e., the nuclear Overhauser effect), the relatively strong spin-spin coupling of adjacent 13C nuclei interferes with the desired observation.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor
  • Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor
  • Site-specific isotopically-labeled proteins, amino acids, and biochemical precursors therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Uniformly 13C-Enriched Catalytic Domain of Human Stromelysin (SCD)

[0044] The 81-256 fragment (SEQ ID NO: 1) of stromelysin (SCD) is prepared by inserting a plasmid which codes for the production of the protein fragment into an E. coli strain and growing the genetically-modified bacterial strain in a suitable culture medium. The protein fragment is isolated from the culture medium, purified, and subsequently used in the two-dimensional NMR analysis of its affinity with test compounds in accordance with the method of this invention. The procedures for the preparation processes are described below.

[0045] Human skin fibroblasts (ATCC No. CRL 1507) are grown and induced using the procedure described by Clark, et al., Archiv. Biochem. and Biophys., 241:36 (1985). Total RNA is isolated from 1 g of cells using a RNAgents® Total RNA Isolation System Kit (Promega Corp., 2800 Woods Hollow. Road, Madison, Wis. 53711, USA) following the manufacturer's instructions. A 1 μg portio...

example 2

Preparation of Specifically 13C-Enriched Catalytic Domain of Human Stromelysin (SCD)

[0056] SCD is expressed by culturing the BL21(DE3) / pLysS / pETST-255-1 modified E. coli strain in a medium comprising 2-keto-4-(13C)-butyric acid, or a salt thereof, and 2-keto-3-(13C-methyl)-4-(13C) -butyric acid, or a salt thereof. The methods used for preparation of the genetically-engineered strain of E. coli, and for expressing, isolating, and purifying the protein fragment are as described above, except for the use of U-12C-glucose, instead of U-13C-glucose.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
optical densityaaaaaaaaaa
pHaaaaaaaaaa
optical densityaaaaaaaaaa
Login to View More

Abstract

Site-specific isotopically-labeled valine, leucine, and isoleucine and biosynthetic precursors for these amino acids are provided. The amino acids are labeled with 13C or 14C at the methyl group carbon atom(s) most remote from the carboxyl group. Also disclosed are the biochemical precursors of these labeled amino acids, 2-keto-4-(nC) butyric acid and 2-keto-3-(nC-methyl)-4-(nC)-butyric acid in which n, at each occurrence, is 13 or 14. Also disclosed are proteins, protein fragments, and polypeptides containing these site-specifically isotopically labeled amino acids, and methods for preparing the biochemical precursors, the amino acids, and the proteins, protein fragments, and polypeptides.

Description

CONTINUING DATA [0001] This application is an original conversion from the provisional application Ser. No. 60 / 128,668, filed Apr. 9, 1999.FIELD OF THE INVENTION [0002] The present invention relates to site-specific isotopically-labeled organic compounds and processes for their preparation. More particularly, the present invention concerns site-specific isotopically-labeled biochemical precursors of leucine, isoleucine, and valine, the isotopically-labeled amino acids per se, proteins, protein fragments or polypeptides made therefrom, and related methods of preparation. BACKGROUND OF THE INVENTION [0003] A recently-developed technique for discovering new drug leads involves the use of nuclear magnetic resonance (NMR) spectroscopy to discover compounds that bind to a particular target molecule such as a protein (.see, for example, U.S. Pat. Nos. 5;698,401 and 5,804,390, to Fesik, et al.). The technique involves the determination of a first two-dimensional 15N / 1H NMR correlation spect...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityApplications(United States)
IPC IPC(8): A61K38/16A61K31/198C12P21/06C07C59/347C07C229/08C12P13/04C12P21/02G01N33/68
CPCC07B2200/05C07C59/347C07C229/08G01N2458/00C12P21/02G01N33/6803C12P13/04
InventorFESIK, STEPHENAUGERI, DAVID
OwnerFESIK STEPHEN