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Device and method for the determination of protein domain boundaries

a technology of protein domain boundaries and devices, applied in the direction of peptide preparation methods, biochemistry apparatus and processes, biochemistry apparatus, etc., can solve the problems of difficult to recover remaining active enzymes for repeated use after the reaction, difficult to obtain suitable crystals, and high labor intensity

Inactive Publication Date: 2003-06-05
AFFINIUM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020] Another advantage is the ability to re-use the treated plates. Another advantage is the reproducibility and as mentioned, high throughput of protease digestion.
[0021] Because many devices can be generated at the same time, there is an efficiency of production and consistency of immobilized protease concentration that can be attained by this invention that is not currently available in the art. Some of the plates with an immobilized protease can be mass produced and stored for at least one week, at 4.degree. C., while maintaining activity. Plates can be manufactured as standards or as custom models as requested.

Problems solved by technology

The limiting step in the technique involves the growth of a suitable crystalline sample.
Because of the complexity of proteins, obtaining suitable crystals can be quite difficult.
This process is extremely time consuming and labour intensive.
However, the fact that the enzyme reaction is a homogeneous reaction in an aqueous solution is a great hindrance to performance of a continuous reaction in industrial applications and also makes it very difficult to recover remaining active enzymes for repeated use after the reaction.
In addition, complicated operational procedures are necessary for separation and purification of the reaction product.
A limitation of the aforementioned strategy is the time consuming nature of cleaving, identifying and isolating the domains of a protein from the digestion solution.
Many commercial automated apparatuses are available and save considerable hours and days of sample manipulation.
Moreover, given the existence of a multitude of known proteases and the application of recombinant DNA technology to the study and production of protease analogs, the art has yet to develop completely.

Method used

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  • Device and method for the determination of protein domain boundaries
  • Device and method for the determination of protein domain boundaries

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0067] General Experimental Protocol for Immobilizing Proteases on Microtiter Plates

[0068] In this example, four proteolytic enzymes are immobilized on a microtiter plate.

[0069] Reagents

[0070] The reagents were prepared as follows. A buffer solution of TBS, 50 mM Tris pH 8.0, 150 mM NaCl is prepared for use with the remaining solutions. A solution of TBS, 0.01% beta-octa-glucoside is prepared as a blocking buffer. The following proteases are prepared: Chymotrypsin, 0.5 mg / ml (in TBS); Trypsin, 0.5 mg / ml (in TBS); Papain, 0.5 mg / ml (in TBS); and Proteinase K, 0.5 mg / ml (in TBS). The protein to be digested is prepared at 65 .mu.g / ml (diluted in TBS).

[0071] Preparation of Immobilized Protease Plate

[0072] In this example of device preparation, Nunclon surface 96 well microtiter plates are used. The proteases are immobilized onto the plates by aliquotting 45 .mu.l TBS into each well. Six wells are used for each protease to generate a series for each protease, each series operating at dec...

example ii

[0080] Comparison of Immobilized Protease Digestion to Solution Protease Digestion

[0081] The protease plates were used to digest five different proteins whose proteolytic digestion pattern in solution was already known. The pattern of digestion for the immobilized proteases mimicked the solution digestion pattern. Thus, we have shown that the immobilized proteases plates could be used to identify stable domains of several different proteins. Shown in FIG. 1 is an example of a limited digestion of the transcription factor TFIIS from the yeast, Saccharomyces cerevisiae. The five lanes shown correspond to the concentration of proteases used (as discussed above in Example I). FIG. 2 illustrates results of digestion on immobilized protease plates of RNA polymerase B5 (see A); RNA polymerase B6 (B); RNA polymerase B8 (C); and D is a bacterial protein parB. Each of these four proteins were digestd by trypsin (panels on the left) and chymotrypsin (panels on the right). These digests compare...

example 111

[0082] Limited Proteolysis

[0083] Four different proteases, trypsin, chymotrypsin, papain and proteinase K (Sigma) were immobilized on plastic 96-well microtitre plates (Nuclon) in the following manner. The protease stocks were made 0.5 mg / ml in TBS (50 mM Tris pH 8, 150 mM NaCl). A serial dilution of each protease was prepared to final concentrations of 50 .mu.g / ml, 25 .mu.g / ml, 5 .mu.g / ml, 2.5 .mu.g / ml and 0.5 .mu.g / ml in TBS. 50 .mu.l of each dilution was applied to different wells in a row of the microtitre plate. The plate with the arrayed protease dilutions was then incubated overnight at 4.degree. C. in a sealed bag containing a wet paper towel.

[0084] The protease solution was then removed and the wells washed with 100 .mu.l of blocking buffer (TBS, 0.01% beta-octyl glucoside). The first wash was discarded and the non-specific binding sites on the microtitre wells were blocked with an additional 30 minute incubation at 4.degree. C. with an additional 100 .mu.l of blocking buff...

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Abstract

A method and device to assist in the determination of protein domain boundaries is described. In particular the device is designed to provide a high throughput of proteolytic digestion of proteins to identify domains and their boundaries, for use in protein structure determination, in a manner that is amenable to automation. Proteases are immobilized in a convenient format such as a microtitre plate and preferably arranged in a matrix thereby allowing for simultaneous degradation of a protein by a number of proteases at a number of concentrations.

Description

[0001] The present invention generally relates to methods for determination of protein structure and in particular concerns the use of immobilized proteases to identify domain boundaries of proteins, and an apparatus for digesting proteins.BACKGROUND OF INVENTION[0002] The molecular structure of proteins allows them play crucial roles in virtually all biological processes, including: enzymatic catalysis, transport and storage; coordinated motion; mechanical support; immune protection; generation and transmission of nerve impulses; and control of growth and differentiation. In particular, the side chains of the different amino acids that comprise proteins, enables these long macromolecules to fold into distinctive structures and form complementary surfaces and clefts and enables them to specifically recognize and interact with highly diverse molecules. The catalytic power of enzymes comes from their capacity to bind substrates in precise orientations and to stabilize transition state...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K1/12C12Q1/37G01N33/68
CPCC07K1/12C12Q1/37Y10S435/97G01N33/6803Y10S435/975G01N33/68
Inventor EDWARDS, ALED
Owner AFFINIUM PHARMA
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