Design, synthesis, and application of a Protein A mimetic

Abstract

Low-molecular-weight synthetic molecules that mimic the activity of native biological macromolecules have therapeutic potential, utility in large-scale production of biopharmaceuticals, and the capacity to act as probes to study molecular recognition events. We have developed a nonpeptidyl mimic for Staphylococcus aureus Protein A (SpA). The specific recognition and complexation elements between the B domain (Fb) of SpA and the Fc fragment of IgG were identified from the x-ray crystallographic structure. Computer-aided molecular modeling was used to design a series of biomimetic molecules around the Phe132-Tyr133 dipeptide involved in its binding to IgG. One of the ligands binds IgG competitively with SpA in solution and when immobilized on agarose beads, with an affinity constant of 10(5)-10(6) M-1. The immobilized artificial Protein A was used to purify IgG from human plasma and murine IgG from ascites fluid, and to remove bovine IgG from fetal calf serum.

One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

“…To better evaluate the chromatographic behavior of the adsorbents, two elution systems (KCl and potassium phosphate) were examined. As already discussed in Section 3.3, adsorbents having no purine moiety (Table 1, entries 1-7) or only purine moieties (Table 1, entries 8,9,[24][25][26] showed an unsatisfactory purifying ability and/or enzyme recovery (yield). All other immobilized ligands displayed variable chromatographic behavior depending on the elution system used (Figs.…”

“…5, in the absence and in the presence of ATP. The calculated dissociation constant was determined to be 0.12 mM in the absence of ATP [24]. It appears that ATP is able to perturb the enzyme-mABSGu complex, since the immobilized ligand has failed to adsorb Taq Pol in the presence of ATP (15 mM).…”

Separation of antigens and antibodies by immunoaffinity chromatography

“…Based on the principle that the dipeptide motif Phe-132:Tyr-133 plays an important role in the interaction of protein A with Fc portion of IgGs, corresponding synthesis of mimetic peptides has been paid attention to. For example, Li et al (1998) have designed and synthesized several molecules to mimic the Phe-132:Tyr-133 dipeptide by using 1,3,5-trichloro-triazine as the scaffold. Two synthetic, nonpeptidyl protein A mimetic ligands − Mab sorbent A1P and A2P were investigated to be used in the initial purification step for capturing of polyclonal antibodies (Ghose et al, 2006).…”

The Improvement of LC-MS/MS Proteomic Detection with Biomimetic Affinity Fractionation

“…As a new affinity fraction, biomimetic chromatography uses synthetic affinity ligands for matrix, which could mimic the properties of natural ligands and circumvent these drawbacks of natural ligands by imparting resistance to chemical and biochemical degradation, displaying ease and low cost of production, and withstanding harsh sterilization without loss of performance (Lowe, 2001). It has been verified that many specific synthetic affinity ligands could be designed to purify different single proteins in our lab and other labs (Li et al, 1998;Teng et al, 1999;Gupta and Lowe, 2004;Melissis et al, 2007;Xin et al, 2007;Dong et al, 2008a). Our lab has constructed affinity ligand library (thousands of ligands), and different ligands showed different absorbance effect to proteins such as purification, depletion of high abundance proteins, and enrichment of low abundance proteins (Dong et al, 2008b), which gives a imaginable application prospect in proteomics.…”