Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Subtilases and subtilase variants having altered immunogenicity

A Bacillus subtilis and variant technology, applied in the direction of enzymes, bacteria, hydrolytic enzymes, etc., can solve the problems of epitope loss and importance reduction

Inactive Publication Date: 2009-08-26
NOVOZYMES AS
View PDF93 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may lead to a reduction in the importance of the epitope, possibly switching it from a high-affinity epitope to a low-affinity epitope, or may even result in the loss of the epitope, i.e., the epitope is not sufficient to bind the antibody to elicit an immune response

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
  • Subtilases and subtilase variants having altered immunogenicity
  • Subtilases and subtilase variants having altered immunogenicity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0310] Identification of epitope sequence and epitope pattern in Savinase

[0311] Epitope sequences and patterns were detected as previously described in Example 1 of WO 01 / 83559.

[0312] From highly diverse phage libraries expressing random hexapeptides, nonapeptides, or dodecapeptides as part of membrane proteins (10 12 ) were screened for their ability to bind purified specific rabbit IgG, and purified rat and mouse IgG1 and IgE antibodies. Phage libraries were obtained according to the prior art (see WO 9215679, which is hereby incorporated by reference).

[0313] Antibodies were raised in corresponding animals by subcutaneous, intradermal or intratracheal injection of selected target proteins (N=75) including Savinase and other subtilases in phosphate buffered saline (PBS). Purified from the sera of immunized animals by affinity chromatography using paramagnetic immunobeads (Dynal AS) loaded with porcine anti-rabbit IgG, mouse anti-rat IgG1 or IgE, or rat anti-mouse...

Embodiment 2

[0324] The location of the amino acid positions involved in the potential IgE epitope on the three-dimensional structure of Savinase

[0325] Use appropriate software (such as SwissPort Pdb Viewer, WebLite Viewer) to manually locate the most likely amino acid positions found in potential IgE epitopes (usually these amino acids are found to be likely to involve at least 3 IgE epitopes) On the three-dimensional structure of Savinase (Protein Data Bank accession number 1SVN; Betzel, C., Klupsch, S., Papendorf, G., Hastrup, S., Branner, S., Wilson, K.S.: alkaline protease from Bacillus lentus Crystal structure of Savinase at 1.4 Å resolution (Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 Å resolution, J Mol Biol 223 p. 427 (1992)).

[0326] By mapping the amino acids on the three-dimensional structure, it was found that the amino acids that may be involved in the IgE epitope clustered in 3 main regions:

[0327] ●Area 1: P14, A15, R19, G20, T...

Embodiment 3

[0332] Position the amino acid positions selected by protein engineering on the three-dimensional structure of Savinase

[0333] Amino acids for epitope protein engineering are selected based on considerations related to structure and enzyme activity, which means preferential selection of those suggested by 3D analysis or empirically from other protein engineering designs to have a beneficial effect on enzyme activity and / or stability Location.

[0334] Selected amino acids are in the following regions:

[0335] ●Area 1: A15, R19, R275

[0336] ●Area 2: S57

[0337] ●Area 3: E136, N140, Y167, R170, A172, D181, R186, A194, G195, R247, T260, L262

[0338] ●Location N218

[0339] These positions are artificially transformed individually or in combination with each other. Positional unions are selected based on the performance of individual mutations and / or topographical patterns (covering as large a region as possible with as few mutations as possible).

[0340] Based on ...

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
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

The present invention relates to subtilase variants and subtilases with altered immunogenicity, in particular subtilase variants and subtilases with reduced allergenicity. The subtilase variant has modifications at position 57 and at least one of positions 170, 181 and 247. The position numbers used refer to the position of subtilisin Novo (BPNAE) from Bacillus amyloliquefaciens. Furthermore, the present invention relates to the expression of said subtilase variants and subtilases and their use in, for example, detergents and oral care products.

Description

field of invention [0001] The present invention relates to subtilases and subtilase variants having altered immunogenicity and uses thereof, and to methods of producing said subtilases and subtilase variants. Background of the invention [0002] More and more proteins, including enzymes, are being produced industrially and used in various industries, housekeeping and medicine. As proteins, they are likely to stimulate immune responses in humans and animals, such as allergies. [0003] Various attempts have been made to alter the immunogenicity of proteins. Usually this change is restricted to only the part of the protein responsible for inducing an immune response, ie, the epitope. An epitope consists of amino acids that may be contiguous in the primary sequence, but more often are located adjacent to each other in the three-dimensional structure of the protein. It has been found that small changes in the epitope may affect its binding to the antibody. This may lead to a...

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 & Authority Patents(China)
IPC IPC(8): C12N9/56C12N15/09A61K8/66A61K8/72C11D3/386C12N1/15C12N1/19C12N1/21C12N5/10C12N9/54
CPCC12N9/54C11D3/386
Inventor E·L·罗根N·T·尼尔松S·恩斯特C·安德森N·W·贝格
Owner NOVOZYMES AS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com