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Controlled dissolution crosslinked protein crystals

a crosslinked protein and crystal technology, applied in the direction of oxidoreductases, enzyme stabilisation, immobilised enzymes, etc., can solve the problems of poor stability of proteins, variability of performance or high cost, and limited us

Inactive Publication Date: 2006-05-18
ALTHEA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment surrounding said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate.

Problems solved by technology

At times, such uses have been limited by constraints inherent in proteins themselves or imposed by the environment or media in which they are used.
Such constraints may result in poor stability of the proteins, variability of performance or high cost.
In the past, environmental elements have often posed barriers to the widespread use of proteins.
However, these approaches have incurred either loss of protein activity or the additional expense of protein stabilizing carriers or formulations.
The objective of controlled release of proteins, however, must be balanced with the fact that the protein itself may not be stable under storage conditions.
Protein stability may also be adversely affected by other components of the formulation in which it is contained.
For example, heavy duty liquid detergents constitute hostile environments for component enzymes.
Such problems have been approached through the use of mutant subtilisin proteases, which are said to have improved oxidative stability.

Method used

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  • Controlled dissolution crosslinked protein crystals
  • Controlled dissolution crosslinked protein crystals
  • Controlled dissolution crosslinked protein crystals

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Crosslinked Subtilisin Crystals

Crystallization of Subtilisin

[0115] One volume of Alcalase 2.5 L (Novo Nordisk Bioindustrials, Franklinton, N.C.) was added to 2 volumes of a solution of 15% sodium sulfate (pH 5.5) prepared at 30-35° C. The crystallization solution was seeded with 1 / 2,000-1 / 500 volume seeds (30 mg / ml slurry of crystals in 15% sodium sulfate (pH 5.5), pH supported at 5.5 by adding NaOH. The seeded crystallization solution was incubated at 30-35° C., stirring by magnetic stirrer overnight. This yielded 60-80% (by activity) crystal rods, 10-50 μm, in length, 1-3 μm in width, after 24-48 hours.

example 2

Crosslinking of Subtilisin Crystals

[0116] Subtilisin crystals were crosslinked using one of a variety of crosslinkers, including: glutaraldehyde, glyoxal, succinaldehyde, octanedialdehyde and epoxides.

Glutaraldehyde Crosslinking

[0117] Glutaraldehyde (“GA”) (supplied as 50% in aqueous by Aldrich Chemical Co.) was diluted in deionized water at 4° C. in the various amounts listed in Table I below. For each ml of subtilisin crystals (27 mg / ml) in 15% sodium sulfate, 10 μl of the diluted glutaraldehyde was added to the slurry while shaking on a vortex at low speed (for amounts less than 5 ml) or stirring with an overhead stirrer at medium speed (for amounts 25 ml-500 ml). After mixing for the allotted crosslinking time, the samples were centrifuged for 20 seconds at maximum speed, the supernatant was discarded and replaced with 15% sodium sulfate. This “washing” was repeated a total of 5 times. The final resuspension was effected with 900 μl of 15% sodium sulfate.

TABLE IGlutaraldeh...

example 3

Activity Assay

[0136] In order to test the activity of crosslinked protein crystals according to this invention, as well as other enzyme samples, we developed the following azocasein assay.

[0137] Materials:

[0138] 2.0M Tris Buffer. 500 ppm CaCl2

[0139] 0.2M Tris Buffer. 50 ppm CaCl2

[0140] 50% urea

[0141] Azocasein

[0142] 5% trichloroacetic acid (“TCA”)

[0143] Alcalase (2.5 L)

[0144] ChiroCLEC-BL™ (crosslinked subtilisin crystals, available from Altus Biologics, Inc., Cambridge, Mass.)

[0145] The assay was carried out, preparing azocasein just prior to use, by dissolving 600 mg azocasein with 10 ml of 50% urea and vortexing lightly to complete the dissolution. Then 10 ml 2.0M Tris was added and vortexed to mix, increasing the volume to 100 ml by adding deionized water.

[0146] The stock solutions of the enzyme to be assayed in 0.2M Tris were prepared, to provide 50 μl aliquots to be assayed, as follows:

Without detergent: 0.03 mg / ml Alcalase (soluble, uncrosslinked subtilisin Carls...

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Abstract

The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment of said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations for decontamination.

Description

TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to crosslinked protein crystals characterized by the ability to change from insoluble and stable form to soluble and active form upon a change in the environment surrounding said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. According to one embodiment of this invention, such crosslinked protein crystals are capable of releasing their protein activity at a controlled rate. This invention also provides methods for producing such crosslinked protein crystals, methods using them for protein delivery and methods using them in cleaning agents, including detergents, pharmaceutical compositions, vaccines, personal care compositions, including cosmetics, veterinary compositions, foods, feeds, diagnostics and formulations fo...

Claims

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Application Information

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IPC IPC(8): A61K8/66C11D3/386C12N9/00C12N9/20A23K1/16A61K8/35A61K8/36A61K8/42A61K8/46A61K8/64A61K8/72A61K9/22A61P31/12A61Q5/02A61Q19/00C07K1/14C07K14/765C11D3/37C11D17/00C12N9/56C12N11/00C12N11/04C12S99/00
CPCA23K1/1631A61K8/64A61Q5/02A61Q19/00C07K14/765C07K2299/00C11D3/3719C11D3/38C12N9/20C12N9/54C12N9/92C12N9/96C12N11/00C12Y304/21062C12Y304/24027C12Y503/01005C30B29/58Y10S530/81A23K20/147A61P31/12A61P43/00C11D3/386
Inventor MARGOLIN, ALEXEY L.PERSICHETTI, ROSE A.ST. CLAIR, NANCY L.KHALAF, NAZER K.
Owner ALTHEA TECH
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