One of the most common antibody purification techniques used SpA immobilized on various solid supports that can be used in industrial application. Changes in SpA immobility methods for improving antibody extraction is still ongoing (Rigi, G. et al. 2019).
Increasing stability of the SPA protein in various harsh environment is one of the main factors in designing of IgG screening (Gerald Cherf and Jennifer R. 2015). One of the major strategies to increase proteins stability is display of proteins on the surface of a live cell as an alternative to classic protein immobilization approach (Mateja Lozancic, et al. 2019; Yadveer S. Grewal, et al. 2016). Classic immobilization of recombinant proteins onto the surface of a matrix (Khodaei, S., 2018) is sometimes challenging as the protein might lose its conformation and consequently its function. However, anchoring of the proteins is a mild approach to immobilize the heterologous proteins to the cell outer membranes. In this way the host cell produces the heterologous protein and at the same time covalently attach it on its surface. In this article, we have used the E.coli surface displaying method to express SPA protein for IgG separation. The immunoadsorbent generated from E. coli surface display, in addition to the other benefits mentioned, can be rapidly generated in a cost-effective way and stored lyophilized at room temperature, which will be stable for several months that reduces the cost of downstream processes in the industry as well.
The efficiency of surface display systems and the correct and efficient protein folding and its stability is highly related to the specifications of the carrier protein, the passenger protein, and the fusion method (Yang, Z., et al. 2008 ; Barrett, T., et al. 2019). LPP-ompA is an efficient surface display system developed and applied for various applications (Fasehee, H., et al. 2018; Rigi, G., et al. 2014 ;Tafakori, V., et al. 2012). In this research project for applying Lpp’-OmpA as an anchor for SPA, in the initial design, we first performed physico-chemical and structural studies on the fusion chimeric protein using molecular dynamics tools to ensure the strength and stability of this novel structure on the cell surface.
Computational analysis showed that the surface expression of SpA295 creates a stable structure and does not form undesirable bonds with the surface protein Lpp’-OmpA, and maintain its binding structure to IgGFc. Furthermore, the analysis displayed in the binding of Lpp’-OmpA-SpA297 complex with IgGFc which aminoacids involve in Vander Waals interaction, hydrogen binding and ionic binding.
In the experimental work, surface expression of this novel system of the recombinant protein by five repeat domains of protein A on the surface of Escherichia coli BL21 and the power of IgG separation confirmed computer simulation findings. The amount of uptake under five SpA repeat domains system was extremely high. The higher IgG-binding yield demonstrated through SDS-PAGE analysis which can be used as a good replacement for the conventional immune precipitates.
The system used in our work, is shown to be a consistent and convenient tool for engineering E. coli cell-surface to display various ligands in their active form. An advantage of the present system to the conventional commercially available immobilization matrices is simplicity, high production rates, easier production and handling processes, and the lower cost of preparing the matrix.
Our matrix was able to purify IgG from human sera as a functional assay and when taking into consideration the yield, purity of IgG and the cost of production of matrix, the system can be used to develop an efficient immunoadsorbent.
We showed that the displayed protein domains with distinct functions of IgG purification at the cellular surface are accessible for the binding partner in binding studies. Furthermore, with the whole cell as a matrix, the proteins have proven to be more stable, therefore making downstream processes of associated preparations and protein purification redundant.
Further investigation on this system is required to achieve higher production rates and specificity at the industrial scale.