Expression, purification and characterization of Protein A for antibody (IgG) detection in rapid diagnostic tests

Abstract

Protein A which is a Staphylococcus aureus cell wall component specifically binds to the Fc region of immunoglobulin G₁ from various species such as humans, rabbits and guinea pigs. The protein serves as one of the bacterium’s virulence factors to evade the host immune system and initiate infection. The outermost part of protein A contains five binding domains that strongly interact with the Fc region of IgG₁, hence protein A has been the most widely used ligand for affinity purification of IgG antibodies. Taking advantage of the high interaction between protein A and IgG, the development of a recombinant protein A for IgG detection in rapid tests seems logical. Expression of protein A has been previously investigated, however, little progress has been made with protein A being found to generate inclusion bodies in prokaryotic systems (Escherichia coli) lowering its solubility. The successful expression and purification of insoluble proteins from inclusion bodies have been difficult tasks to conduct. Therefore, this study aimed to express and purify a soluble protein A from inclusion bodies through Immobilized Metal Affinity Chromatography (IMAC) and asses its binding capabilities to IgG antibody by Enzyme-Linked Immunosorbent Assays (ELISA) and Isothermal Titration Calorimetry (ITC) for its application in Lateral Flow Devices. In this study, recombinant protein A was expressed in T7 Express Competent E. coli (High Efficiency) bacterial cells and purified though IMAC. Enzyme-Linked Immunosorbent Assays were conducted to analyze the binding capabilities of the purified recombinant protein A to IgG antibodies. The secondary structural determination of protein A was predicted by an online bioinformatics tool (Self-Optimized Prediction Method Alignment (SOPMA)) and further determined with Circular Dichroism (CD) spectroscopy. ExPASy ProtParam tool was used to predict the number of chromophores in the protein and the tertiary structure of the protein was evaluated by far-UV (Ultra Violet) fluorescence. The binding energetics of IgG antibody to protein A were determined by ITC. The purified recombinant protein A was further tested in the lateral flow assays to detect its binding abilities to gold nanoparticles and also if it can bind to HIV antibodies applied. One milligram of purified recombinant protein A was obtained from 600 mL of bacterial culture. The purified protein A showed a stronger binding affinity to Rabbit anti-Mouse IgG than Goat anti-Rabbit IgG antibody and as expected protein A showed a very low binding affinity to IgM antibody. The secondary structure prediction with SOPMA resulted in the prediction of 48.62% alpha helix, 40.75% random coil and 17% beta-sheet. The CD spectrum obtained for the recombinant protein A showed a negative peak at 200 nm, characteristic of a predominantly random coil protein. Through ExPASy ProtParam online tool, the protein was predicted to be composed of 0% tryptophan, 1.2% tyrosine and 3.1% phenylalanine. The binding energetics of Rabbit anti-Mouse IgG to the purified protein A through ITC proved to be enthalpically driven, with ΔH value of -100kJ/mol, indicating an exothermic reaction. The protein showed good binding abilities to gold nanoparticles as it was able to bind/molecular recognize the HIV antibody applied. The ability of the recombinant protein A to be able to recognize and form a conjugated pair with the gold nanoparticles and also its ability to capture the HIV antigens and the HIV antibodies in the lateral flow assay further confirmed the functionality of the protein.