Engineering streptococcal protein G for increased alkaline stability.


Abstract

Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.

Submission Details

ID: adGYZiDN4

Submitter: Shu-Ching Ou

Submission Date: Nov. 16, 2018, 1:54 p.m.

Version: 1

Publication Details
Gülich S;Linhult M;Ståhl S;Hober S,Protein Eng (2002) Engineering streptococcal protein G for increased alkaline stability. PMID:12468718
Additional Information

Study Summary

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
4HAF 2012-09-26T00:00:00+0000 2.04 Crystal structure of fc-fragment of human IgG2 antibody (primitive crystal form)
4HAG 2012-09-26T00:00:00+0000 3.4 Crystal structure of fc-fragment of human IgG2 antibody (centered crystal form)
4L4J 2013-06-07T00:00:00+0000 1.92 Crystal structure of fc-fragment of human IgG2-Sigma antibody
7LUS 2021-02-23T00:00:00+0000 2.45 IgG2 Fc Charge Pair Mutation version 1 (CPMv1)
7LUS 2021-02-23T00:00:00+0000 2.45 IgG2 Fc Charge Pair Mutation version 1 (CPMv1)
4WWI 2014-11-11T00:00:00+0000 2.31 Crystal structure of the C domain of staphylococcal protein A in complex with the Fc fragment of human IgG at 2.3 Angstrom resolution
4ZNC 2015-05-04T00:00:00+0000 2.28 Fc fragment of human IgG in complex with the C domain of staphylococcal protein A mutant - Q9W
5W38 2017-06-07T00:00:00+0000 1.8 1.80A resolution structure of human IgG3 Fc (N392K)
6D58 2018-04-19T00:00:00+0000 2.39 Crystal structure of a Fc fragment of Human IgG3
1ADQ 1997-02-18T00:00:00+0000 3.15 CRYSTAL STRUCTURE OF A HUMAN IGM RHEUMATOID FACTOR FAB IN COMPLEX WITH ITS AUTOANTIGEN IGG FC

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
192.8 C,D Immunoglobulin heavy constant gamma 1 P06654 SPG1_STRSG
200.0 C,D Immunoglobulin heavy constant gamma 1 P19909 SPG2_STRSG
185.4 A,B Immunoglobulin heavy constant gamma 1 P01860 IGHG3_HUMAN
186.4 A,B Immunoglobulin heavy constant gamma 1 P01861 IGHG4_HUMAN
187.4 A,B Immunoglobulin heavy constant gamma 1 P01859 IGHG2_HUMAN
193.2 A,B Immunoglobulin heavy constant gamma 1 P01857 IGHG1_HUMAN
193.2 A,B Immunoglobulin heavy constant gamma 1 P0DOX5 IGG1_HUMAN