Molecular Modification of Protein A to Improve the Elution pH and Alkali Resistance in Affinity Chromatography

Abstract

Protein A of Staphylococcus aureus has been widely used as an affinity ligand for the purification of immunoglobulin. However, the low elution pH and the sensitivity to alkaline condition restricted the large-scale application of antibody purification. To overcome these disadvantages, the B domain was selected and mutated to Z domain and the recombinant Protein A was reconstructed by linking five Z domains. First, a section of six glycines was inserted into the second loop of Z domain, Z (6G). This increased the elution pH to 4.0-5.0. Then, the site-specific mutagenesis was conducted by replacing the 23rd asparagines to threonine and 30th phenylalanine to alanine, Z (N23T, F30A). These mutations made the recombinant Protein A shown a higher alkaline resistance than the nature Protein A. The work confirmed the modification of Protein A and exhibited the characteristics of recombinant Staphylococcal Protein A for antibody purification.

Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

“…To address the issue with CIP procedures, SpA was shown to have higher alkaline stability with a single mutation at position 29, with G29W being the most stable (142). Two further mutations N23T and F30A to the SpA Z domain resulted in a higher alkaline resistance when compared to wild type (143). SpA Z domain was also engineered to include six glycine residues on the second loop, which resulted in an increase in the elution pH (143).…”

“…Two further mutations N23T and F30A to the SpA Z domain resulted in a higher alkaline resistance when compared to wild type (143). SpA Z domain was also engineered to include six glycine residues on the second loop, which resulted in an increase in the elution pH (143). Wild type SpA is less susceptible to extreme alkaline conditions, with a halflife of 16 h (141) compared to SpG, which has a half-life of under 10 mins (144).…”

Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

“…To address the issue with CIP procedures, SpA was shown to have higher alkaline stability with a single mutation at position 29, with G29W being the most stable [138]. Two further mutations N23T and F30A to the SpA Z domain resulted in a higher alkaline resistance when compared to wild type [139]. SpA Z domain was also engineered to include six glycine residues on the second loop, which resulted in an increase in the elution pH [139].…”

“…Two further mutations N23T and F30A to the SpA Z domain resulted in a higher alkaline resistance when compared to wild type [139]. SpA Z domain was also engineered to include six glycine residues on the second loop, which resulted in an increase in the elution pH [139]. Wild type SpA is less susceptible to extreme alkaline conditions, with a half-life of 16 h [137] than SpG, which has a half-life of under 10 mins [140].…”

Rational design of affinity ligands for bioseparation

“…Using a destabilized variant of the Z-domain which does not bind the Fc fragment as a template, Hober and co-workers showed that a N23T mutation increased chemical resistance to withstand cleaning-in-place protocols which is a critical parameter for biological affinity reagents [44]. More recently, Xia et al showed thatthe addition of six glycines in loop 2 of the Z-domain along with mutations in residues N29T and F30A greatly increased alkaline resistance to sodium hydroxide extending cleaning in place (CIP) up to 2M in 1h and allowed recovery of antibodies at milder pH [45]. The approach that conferred the Z-domain alkaline resistance was tested towards the C-domain of SpA.…”