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Mutant protein

a technology of mutant proteins and proteins, applied in the field of mutant proteins, can solve problems such as capacity decline, and achieve the effects of improving cleaning tolerance, improving stability, and increasing ph-values

Inactive Publication Date: 2006-08-31
GE HEALTHCARE BIO SCI CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] One object of the present invention is to provide a mutated immunoglobulin-binding protein ligand that exhibits an improved stability at increased pH-values, and accordingly an improved tolerance to cleaning under alkaline conditions, as compared to the parental molecule.

Problems solved by technology

For many affinity chromatography matrices containing proteinaceous affinity ligands such alkaline environment is a very harsh condition and consequently results in decreased capacities owing to instability of the ligand to the high pH involved.

Method used

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  • Mutant protein
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Results (Example 1)

[0109] All Z variants were successfully produced intracellular in E. coli at 37° C. and show the same expression levels, approximately 50 mg / l as estimated from SDS-PAGE. The proteins were all purified by IgG affinity chromatography. After the purification, samples were analysed with SDS-PAGE (data not shown), lyophilised and stored for further analyses. The molecular mass for protein Z and the different mutants thereof were also confirmed by mass spectrometry. The data confirmed correct amino acid content for all mutants (data not shown). Also, structural analyses were performed on a Circular Dichroism (CD) equipment, since it previously has been proven to be suitable for detecting structural changes in α-helical proteins (Johnson, C. W., Jr. 1990. Protein secondary structure and circular dichroism: a practical guide. Proteins. 7:205-214; and Nord, K., J. Nilsson, B. Nilsson, M. Uhlèn, and P.-Å. Nygren. 1995. A combinatorial library of an a-helical bacterial rece...

example 2

Results (Example 2)

[0111] To determine the differences in affinity for the Z variants towards IgG, surface plasmon resonance (SPR) using a Biacore was carried out. The aim was to compare the affinity for the different mutated Z variants according to the invention with the parental molecule. As mentioned above, due to the high alkaline stability of the parental Z domain it was decided to use a structurally destabilised variant of Z including the F30A mutation (Cedergren, L., R. Andersson, B. Jansson, M. Uhlèn, and B. Nilsson. 1993. Mutational analysis of the interaction between staphylococcal protein A and human IgG1. Protein eng. 6:441-448). Therefore, it was of importance to first confirm that the affinity between the mutated molecule and IgG was retained despite the mutation. As can be seen in table 1 below, the affinity of Z(F30A) is not significantly affected. The very small change in affinity gives a slightly higher stability to the complex of Z(F30A) and IgG compared to the pa...

example 3

Results (Example 3)

[0113] Z, Z(F30A), and mutants thereof were covalently attached to HiTrap™ columns using NHS-chemistry. IgG in excess was loaded and the amount of eluted IgG was measured after each cycle to determine the total capacity of the column. Between each cycle the columns were exposed to CIP treatment consisting of 0.5 M NaOH. After 16 pulses, giving a total exposure time of 7.5 hours, the column with the Z(F30A)-matrix shows a 70% decrease of the capacity. The degradation data in FIG. 2a suggest that four of the exchanged asparagines (N6, N11, N43 and N52) are less sensitive to the alkaline conditions the mutants are exposed for in this experiment. In contrast, N23 seems to be very important for the stability of Z(F30A). Z(F30A,N23T) shows only a 28% decrease of capacity despite the destabilising F30A-mutation. Hence, the Z(F30A,N23T) is almost as stable as Zwt and thereby the most stabilised variant with Z(F30A) as scaffold. Also the Z(F30A)-domain with two additional ...

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Abstract

The present invention relates to an immunoglobulin-binding protein, wherein at least one asparagine residue has been mutated to an amino acid other than glutamine or aspartic acid, which mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental molecule. The protein can for example be derived from a protein capable of binding to other regions of the immunoglobulin molecule than the complementarity determining regions (CDR), such as protein A, and preferably the B-domain of Staphylococcal protein A. The invention also relates to a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support, in which protein ligand at least one asparagine residue has been mutated to an amino acid other than glutamine.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10 / 508,625 filed Sep. 20, 2004, which is a filing under 37 U.S.C. § 371 and claims priority to international patent application number PCT / SE03 / 00475 filed Mar. 20, 2003, published on Oct. 2, 2003 as WO03 / 080655 and also claims priority to patent application number 0200943-9 filed in Sweden on Mar. 25, 2002; the disclosures of which are incorporated herein by reference in their entireties.TECHNICAL FIELD [0002] The present invention relates to the field of mutant proteins, and more specifically to a mutant protein that exhibits improved stability compared to the parental molecule as well as to a method of producing a mutant protein according to the invention. The invention also relates to an affinity separation matrix, wherein a mutant protein according to the invention is used as an affinity ligand. BACKGROUND OF THE INVENTION [0003] A great number of applic...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/06C12N1/21C07K14/31C12N15/74B01J20/281B01J20/24C07K1/22C07K14/735C07K16/00C07K16/06C07K19/00C12N15/09G01N30/88
CPCB01D15/3809B01J20/286B01J20/3244C07K1/22C07K14/31C07K16/065C07K14/195Y10S530/81Y10S530/825B01J20/24B01J20/289B01J2220/44B01J2220/52C07K14/001
Inventor HOBER, SOPHIAJOHANSSON, HANS
Owner GE HEALTHCARE BIO SCI CORP
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